Living his entire adult life in Baltimore, Hopkins made many friends among the city's social elite, many of them Quakers. One of these friends was George Peabody, who was also born in 1795, and who in 1857 founded the Peabody Institute in Baltimore. Other examples of public giving were evident in the city, as public buildings housing free libraries, schools and foundations sprang up along the city's widening streets. On the advice of Peabody, some believe, Hopkins determined to use his great wealth for the public good.

Johns Hopkins' Quaker faith and his early life experiences, in particular the 1807 emancipation, had a lasting influence throughout his life and his posthumous legacy as a businessman, railroad man, banker, investor, ship owner, philanthropist and a founder of several Institutions. From very early on, Johns Hopkins had looked upon his wealth as a trust to benefit Future generations. He is said to have told his gardener that, "like the man in the parable, I have had many talents given to me and I feel they are in trust. I shall not bury them but give them to the lads who long for a wider education"; his philosophy quietly anticipated Andrew Carnegie's much publicized Gospel of Wealth by more than 25 years.

Hopkins' early experiences and successes in Business came when he was put in charge of the store while his uncle was away during the War of 1812. After seven years with his uncle, Hopkins went into Business together with Benjamin Moore, a fellow Quaker. The Business partnership was later dissolved with Moore alleging Hopkins' penchant for capital accumulation as the cause for the divide.

After Moore's withdrawal, Hopkins partnered with three of his brothers and established Hopkins & Brothers Wholesalers in 1819. The company prospered by selling various wares in the Shenandoah Valley from Conestoga wagons, sometimes in exchange for corn whiskey, which was then sold in Baltimore as "Hopkins' Best". The bulk of Hopkins' fortune however was made by his judicious Investments in myriad ventures, most notably the Baltimore and Ohio Railroad (B&O), of which he became a Director in 1847 and chairman of the Finance Committee in 1855. He was also President of Merchants' Bank as well as Director of a number of other organizations. After a successful career, Hopkins was able to retire at the age of 52 in 1847.

The Civil War had taken its toll on Baltimore, however, as did the yellow fever and cholera epidemics that repeatedly ravaged the nation's cities, killing 853 in Baltimore in the summer of 1832 alone. Hopkins was keenly aware of the city's need for medical facilities, particularly in light of the medical advances made during the war, and in 1870 he made a will setting aside seven million dollars — mostly in B&O stock — for the incorporation of a free hospital and affiliated medical and nurse's training colleges, as well as an orphanage for colored children and a university. The hospital and orphan asylum would each be overseen by the 12-member hospital board of trustees, and the university by the 12-member university board of trustees. Many board members were on both boards. Johns Hopkins' bequest was used to posthumously found the Johns Hopkins Colored Children Orphan Asylum first as he requested, in 1875; the Johns Hopkins University in 1876; the Johns Hopkins Press, the longest continuously operating academic press in America, in 1878; the Johns Hopkins Hospital and the Johns Hopkins School of Nursing in 1889; the Johns Hopkins University School of Medicine 1893; and the Johns Hopkins School of Hygiene and Public Health in 1916.

A charitable individual, Hopkins put up his own money more than once to not only aid Baltimore City during times of financial crises, but also to twice bail the railroad out of debt, in 1857 and 1873. In 1996, Johns Hopkins ranked 69th in "The Wealthy 100: From Benjamin Franklin to Bill Gates - A Ranking of the richest Americans, Past and Present".

Hopkins' support of Abraham Lincoln also often put him at odds with some of Maryland's most prominent people, particularly Supreme Court Justice Roger B. Taney, who continually opposed Lincoln's presidential decisions, such as his policies of limiting habeas corpus and stationing troops in Maryland. In 1862 Hopkins wrote a letter to Lincoln requesting the President not to heed the detractors' calls and continue to keep Soldiers stationed in Maryland. Hopkins also pledged financial and logistic support to Lincoln, in particular the free use of the B&O railway system.

After the Civil War and during Reconstruction, Johns Hopkins' stance on abolitionism infuriated many prominent people in Baltimore. During the American Reconstruction period to his death his abolitionism was expressed in the documents founding the Johns Hopkins Institutions, and reported in newspaper articles before, during, and after the founding of these institutions. Before the war, there was significant written opposition to his support for Myrtilla Miner's founding of a school for African American females (now the University of the District of Columbia). Similarly, opposition (and some support) was expressed during Reconstruction, such as in 1867, the same year he filed papers incorporating the Johns Hopkins Institutions, when he attempted unsuccessfully to stop the convening of the Maryland Constitutional Convention where the Democratic Party came into power and where a new state Constitution, the Constitution still in effect, was voted to replace the 1864 Constitution of the Radical Republicans previously in power.

Apparent also in the literature of the times was opposition, and support for, the various other ways he expressed opposition to the racial practices that were beginning to emerge, and re-emerge as well, in the city of Baltimore, the state of Maryland, the nation and in the posthumously constructed and founded institutions that would carry his name, A Baltimore American Journalist praised Hopkins for founding three institutions, a university, a hospital and an orphan asylum, specifically for colored children, adding that Hopkins was a "man (beyond his times) who knew no race" citing his provisions for both blacks and whites in the plans for his hospital. The reporter also pointed to similarities between Benjamin Franklin's and Johns Hopkins' views on hospital care and construction, such as their shared interest in free hospitals and the availability of emergency services without prejudice. This article, first published in 1870, also accompanied Hopkins' obituary in the Baltimore American as a tribute in 1873. Cited in many of the newspaper articles on him during his lifetime and immediately after his death were his provisions of scholarships for the poor, and quality health services for the underserved, the poor without regard to their age, sex and color, the colored children asylum and other orphanages, the mentally ill and convalescents.

By the end of Gilman's presidency, Johns Hopkins University, Johns Hopkins Press, Johns Hopkins Hospital and Johns Hopkins School of Nursing, and Johns Hopkins School of Medicine, and Johns Hopkins Colored Children Orphan Asylum had been founded, the latter by the trustees, and the others in the order listed under the Gilman administration. "Sex" and "color" were major issues in the early history of the Johns Hopkins Institutions. The founding of the School of Nursing is usually linked to Johns Hopkins' statements in his March 1873 instruction letter to the trustees that "I Desire you to establish, in connection with the hospital, a training school for female Nurses. This provision will secure the services of women competent to care for those sick in the hospital wards, and will enable you to benefit the whole community by supplying it with a class of trained and experienced nurses".

As per Johns Hopkins' instruction letter, the Johns Hopkins Colored Children Orphan Asylum (JHCCOA) was founded first, in 1875, a year before Gilman's inauguration, now the founding date of the university. The construction of the asylum, including its educational and living facilities, was praised by The Nation and the Baltimore American, the latter stating that the orphan asylum was a place where "nothing was wanting that could benefit science and humanity". As was done for other Johns Hopkins Institutions, it was planned after visits and correspondence with similar institutions in Europe and America.

As he became able, Hopkins provided for his extended family, both during his life and posthumously through his will. He bequeathed a home for Elizabeth, where she lived until her death in 1889.

Women's most well known success after the founding of the nursing school was their requirement that they be allowed to attend Johns Hopkins medical institutions after they provided funds that made possible the opening of the School of Medicine in 1893. Five African American women were among the first women enrolled in the Johns Hopkins University's undergraduate school in 1970. Furthermore, Kelly Miller and Frederick Scott were the first persons of African descent to attend the Johns Hopkins University's graduate and undergraduate schools, respectively. Frederick Scott was the first graduate of African descent from Johns Hopkins University, and he, Robert Gamble, and Kenyan-born James Nabwangu were the first graduates of African descent from Johns Hopkins University's undergraduate school, and Johns Hopkins' medical schools (1967) respectively. Those employees holding jobs in the Service sector are those of African descent who have the longest and most continuous history at the Johns Hopkins Institutions.

The Johns Hopkins Orphan Asylum opened with 24 boys and girls. Under Gilman and his successors, this orphanage was later changed to serve as an orphanage and training school for black female orphans principally as domestic workers, and next as an "orthopedic convalescent" home and school for "colored crippled" children and orphans. The asylum was eventually closed in 1924 nearly fifty years after it opened, and was never reopened.

A biography entitled Johns Hopkins: A Silhouette written by his cousin, Helen Hopkins Thom, was published in 1929 by the Johns Hopkins University Press.

In 1973 Johns Hopkins was cited prominently in the Pulitzer Prize winning book The Americans: The Democratic Experience by Daniel Boorstin, former head of the Library of Congress. From November 14, 1975 to September 6, 1976 a portrait of Hopkins was displayed at the National Portrait Gallery in an exhibit on the democratization of America based on Boorstin's book. In 1989, the United States Postal Service issued a $1 postage stamp in Johns Hopkins' honor, as part of the Great Americans series.