After graduation, White worked for the United Press (now United Press International) and the American Legion News Service in 1921 and 1922. In 1922-23, he was a cub reporter for The Seattle Times. On one occasion, when White was stuck writing a story, a Times Editor said, "Just say the words." He then worked for almost two years with the Frank Seaman advertising agency as a production assistant and copywriter before returning to New York City in 1924. When The New Yorker was founded in 1925, White submitted manuscripts to it. Katharine Angell, the literary Editor, recommended to editor-in-chief and founder Harold Ross that White be hired as a staff Writer. However, it took months to convince him to come to a meeting at the office and additional weeks to convince him to work on the premises. Eventually, he agreed to work in the office on Thursdays.

E. B. White published his first article in The New Yorker in 1925, then joined the staff in 1927 and continued to contribute for almost six decades. Best recognized for his essays and unsigned "Notes and Comment" pieces, he gradually became the magazine's most important contributor, this at a time when it was arguably the most important literary magazine in America. From the beginning to the end of his career at The New Yorker, he frequently provided what the magazine calls "Newsbreaks" (short, witty comments on oddly worded printed items from many sources) under various categories such as "Block That Metaphor." He also served as a columnist for Harper's Magazine from 1938 to 1943.

In 1929, White and Angell were married. They had a son, Joel White, a naval Architect and boat builder, who later owned Brooklin Boat Yard in Brooklin, Maine. Katharine's son from her first marriage, Roger Angell, has spent decades as a fiction Editor for The New Yorker and is well known as the magazine's baseball Writer.

In the late 1930s, White turned his hand to children's fiction on behalf of a niece, Janice Hart White. His first children's book, Stuart Little, was published in 1945, and Charlotte's Web appeared in 1952. Stuart Little initially received a lukewarm welcome from the literary community. However, both books went on to receive high acclaim, and Charlotte's Web won the Newbery Medal from the American Library Association.

In 1949, White published Here Is New York, a short book based on an article he had been commissioned to write for Holiday. That article reflects the writer's appreciation of a city that provides its residents with both "the gift of loneliness and the gift of privacy." It concludes with a dark note touching on the forces that could destroy the city that he loved. This prescient "love letter" to the city was re-published in 1999 on his centennial with an introduction by his stepson, Roger Angell.

In 1959, White edited and updated The Elements of Style. This handbook of grammatical and stylistic guidance for Writers of American English was first written and published in 1918 by william Strunk Jr., one of White's professors at Cornell. White's reworking of the book was extremely well received, and later editions followed in 1972, 1979, and 1999. Maira Kalman illustrated an edition in 2005. That same year, a New York Composer named Nico Muhly premiered a short opera based on the book. The volume is a standard tool for students and Writers and remains required reading in many composition classes. The complete history of The Elements of Style is detailed in Mark Garvey's Stylized: A Slightly Obsessive History of Strunk & White's The Elements of Style.

White received the Laura Ingalls Wilder Medal from the U.S. professional children's librarians in 1970. "substantial and lasting contributions to children's literature." That year he was also the U.S. nominee and eventual runner-up for the biennial Hans Christian Andersen Award, as he was again in 1976. Also in 1970, White's third children's novel was published, The Trumpet of the Swan. In 1973 it won the Sequoyah Award from Oklahoma and the william Allen White Award from Kansas, both selected by students voting for their favorite book of the year. In 2012, School Library Journal sponsored a survey of readers which identified Charlotte's Web as the best children's novel ("fictional title for readers 9–12" years old). The librarian who conducted it said, "It is impossible to conduct a poll of this sort and expect [White's novel] to be anywhere

In 1978, White won a special Pulitzer Prize citing "his letters, essays and the full body of his work". He also received the Presidential Medal of Freedom in 1963 and honorary memberships in a variety of literary societies throughout the United States. The 1973 Oscar-nominated Canadian animated short The Family That Dwelt Apart is narrated by White and is based on his short story of the same name.

White suffered from Alzheimer's disease and died on October 1, 1985, at his farm home in North Brooklin, Maine. He is buried in the Brooklin Cemetery beside Katharine, who died in 1977.