Loïs began her career in textiles after graduating from the School of the Museum of Fine Arts in Boston in 1927. She had already won a number of design competitions and done graduate work. Scholarships were not always color blind; one required that she clean the studio. As a freelance textile designer, she sold designs to F.A. Foster in Boston and Schumacher Company in New York was proud to see them widely used. But one day in 1928, the course of her career changed drastically. She was driving to her family home on Martha's Vineyard and saw one of her designs in the window of an interior decorator's shop in Connecticut. She decided to go in and introduce herself as the fabric designer. Instead of the welcome she expected, the owner said, "How could you have done that? You're a colored girl." At that moment, Loïs realized she needed to produce art for which she would be recognized. She applied for work as an art teacher at the School of the Museum of Fine Arts, her alma mater, where she had won several prizes. Henry Hunt Clarke, the director, told her there were no openings, and perhaps she should think of going down south to "help her people," a suggestion that surprised the middle-class Boston woman.

Although she was denied a teaching job at her Boston alma mater, Loïs went on to a long and distinguished career in art education at Howard University beginning in 1930. Coincidently, her route to Howard began in the south at Palmer Memorial Institute in Sedalia, North Carolina, shortly after her conversation with Henry Hunt Clark. At a seminar, Charlotte Hawkins Brown urged young professionals to come to Palmer and teach. When Loïs offered to start an art department there, Brown turned her down, saying she was too young. Now determined to go south, Loïs persisted and joined the Palmer staff in 1929 on a two-year contract. Like all prep school teachers, she was busy with class assignments and extra-curricular responsibilities, including coaching the women's basketball team and teaching folk dancing. She also found time to paint in Sedalia and a portrait of one of her students, which she called Negro Youth, won honorable mention in the 1930 Harmon Foundation exhibition. In the spring of 1930, James V. Herring, founder of Howard University's art department, came to Palmer at Loïs's invitation to speak to her students and see an exhibit of their work. He was very impressed with Loïs and wanted her to teach at Howard, but she was in the middle of a two-year contract. Herring was confident he could free her from the contract, and whatever negotiations took place, no one knows, but he was successful.

Loïs joined instructors James A. Porter and James L. Wells in the Howard University Department of Art in September 1930 as an instructor of design in the cultural center of Washington, D.C. For her first sabbatical year in 1937, Loïs went to Paris, where there were no color barriers and she felt completely free and equal. Among the people she befriended, learned from, and painted were Émile Bernard, Josephine Baker, Albert Smith, and Céline Tabary. Loïs exhibited her work and received much recognition at the school and in the local press. Bernard considered her a great talent and told her that, unlike so many who painted in Paris, she was not wasting canvas and paint.

In the course of her forty-seven-year career at Howard, she inspired thousands of students with her high standards and encouraging critiques, led workshops and tours which broadened students' minds to more than design and watercolor, and always continued to study and paint.

The last ten years of her life were exciting, challenging, and rewarding for her. She had made a name for herself, and many heads of state, among them President Bill Clinton and French president Jacques Chirac, met her and collected her work. "The World of Loïs Mailou Jones" solo traveling exhibit began a six-year nationwide tour in 1990, including the Corcoran Gallery in Washington, where they held an 89th birthday party for her and apologized for earlier prejudicial policies. She spent every summer painting and exhibiting on her beloved Martha's Vineyard. Bill and Hillary Rodham Clinton collected Breezy Day at Gay Head in 1993 and posed for pictures, and Chelsea Clinton attended the Corcoran party.

Loïs gracefully passed away on June 9, 1998. She is buried in Oak Bluffs cemetery on Martha's Vineyard in a family plot. Her work is displayed in museums around the world and is prized by collectors.