When she was attending Wausau West High School, Mayer was on the curling team and the precision dance team. She excelled in chemistry, calculus, biology, and physics. She took part in extracurricular activities, becoming President of her high school's Spanish club, treasurer of the Key Club, captain of the debate team, and captain of the pom-pom squad. Her high school debate team won the Wisconsin state championship and the pom-pom squad was the state runner-up. During high school, she worked as a grocery clerk. After graduating from high school in 1993, Mayer was selected by Tommy Thompson, then the Governor of Wisconsin, as one of the state's two delegates to attend the National Youth Science Camp in West Virginia.

Intending to become a pediatric neurosurgeon, Mayer took pre-med classes at Stanford University. She later switched her major from pediatric neuroscience to symbolic systems, a major which combined philosophy, cognitive psychology, linguistics, and computer science. At Stanford, she danced in the university ballet's Nutcracker, was a member of parliamentary debate, volunteered at children's hospitals, and helped bring computer science education to Bermuda's schools. During her junior year, she taught a class in symbolic systems, with Eric S. Roberts as her supervisor. The class was so well received by students that Roberts asked Mayer to teach another class over the summer. Mayer went on to graduate with honors from Stanford with a BS in symbolic systems in 1997 and an MS in computer science in 1999. For both degrees, her specialization was in artificial intelligence. For her undergraduate thesis, she built travel-recommendation software that advised users in natural-sounding human language. In 2009, the Illinois Institute of Technology granted Mayer an honoris causa doctorate degree in recognition of her work in the field of search.

After graduating from Stanford, Mayer received 14 job offers, including a teaching job at Carnegie Mellon University and a consulting job at McKinsey & Company. She joined Google in 1999 as employee number 20. She started out writing code and overseeing small teams of Engineers, developing and designing Google's search offerings. She became known for her attention to detail, which helped land her a promotion to product manager, and later she became Director of consumer web products. She oversaw the layout of Google's well-known, unadorned search homepage. She was also on the three-person team responsible for Google AdWords, which is an advertising platform that allows businesses to show their product to relevant potential customers based on their search terms. AdWords helped deliver 96% of the company's revenue in the first quarter of 2011.

In 2002, Mayer started the Associate Product Manager (APM) program, a Google mentorship initiative to recruit new talents and cultivate them for leadership roles. Each year, Mayer selected a number of junior employees for the two-year program, where they took on extracurricular assignments and intensive evening classes. Notable graduates of the program include Bret Taylor and Justin Rosenstein. In 2005, Mayer became Vice President of Search Products and User Experience. Mayer held key roles in Google Search, Google Images, Google News, Google Maps, Google Books, Google Product Search, Google Toolbar, iGoogle, and Gmail.

Mayer is Lutheran, but she has said--referencing Vince Lombardi's "Your God, your family and the Green Bay Packers"--that her priorities are "God, family and Yahoo!, except I'm not that religious, so it's really family and Yahoo!." Since 2008, Mayer has lived on the 38th-floor penthouse suite at the Four Seasons Hotel in San Francisco.

Mayer married Lawyer and investor Zachary Bogue on December 12, 2009. On the day Yahoo! announced her hiring, Mayer revealed that she was pregnant; she gave birth to a baby boy on September 30, 2012. Although she asked for baby name suggestions via social media, she eventually chose the name Macallister from an existing list. On December 10, 2015, Mayer announced that she had given birth to identical twin girls, Marielle and Sylvana.

Mayer was the vice President of Google Search Products and User Experience until the end of 2010, when she was asked by then-CEO Eric Schmidt to head the Local, Maps, and Location Services. In 2011, she secured Google's acquisition of survey site Zagat for $125 million. While Mayer was working at Google, she taught introductory computer programming at Stanford and mentored students at the East Palo Alto Charter School. She was awarded the Centennial Teaching Award and the Forsythe Award from Stanford.

On July 16, 2012, Mayer was appointed President and CEO of Yahoo!, effective the following day. She is also a member of the company's board of Directors. To simplify the bureaucratic process and "make the culture the best version of itself", Mayer launched a new online program called PB&J. It collects employee complaints, as well as their votes on problems in the office; if a Problem generates at least 50 votes, online management automatically investigates the matter. In February 2013, Mayer oversaw a major personnel policy change at Yahoo! that required all remote-working employees to convert to in-office roles. Having worked from home toward the end of her pregnancy, Mayer returned to work after giving birth to a boy, and built a mother's room next to her office suite—Mayer was consequently criticized for the telecommuting ban. In April 2013, Mayer changed Yahoo!'s maternity leave policy, lengthening its time allowance and providing a cash bonus to parents. CNN noted this was in line with other Silicon Valley companies, such as Facebook and Google. Mayer has been criticized for many of her management decisions in pieces by The New York Times and The New Yorker.

Mayer made Fortune magazine history in 2013, as the only person to feature in all three of its annual lists during the same year: Businessperson of the Year (No. 10), Most Powerful Women (at No. 8), and 40 Under 40 (No. 1) at the same time. In March 2016, Fortune then named Mayer as one of the world's most disappointing Leaders.

An earlier lawsuit was filed by Gregory Anderson, who was fired in 2014, alleging the company’s performance management system was arbitrary and unfair and disguised layoffs as terminations for the purpose of evading state and federal WARN Acts, making it the first WARN Act and gender discrimination lawsuit Yahoo! and Mayer faced in 2016.

On 24 December 2015, Mayer was listed by UK-based company Richtopia at number 14 in the list of 500 Most Influential CEOs.

Mayer appeared on the List of women CEOs of Fortune 500 companies in 2017, having ranked 498 of the top 500 Fortune 500 company CEOs.