Born in Nashville, Tennessee, Mary Pennington grew up in Philadelphia, where she often joined her father, a business executive, in his backyard gardening hobby. A library book on medical chemistry revealed Pennington's life direction when she was twelve years old.

In 1892 Pennington completed the requirements for a B.S. degree at the University of Pennsylvania but was not allowed to receive her degree because of her sex. She later earned her Ph.D. from that same university in 1895. After several years of postgraduate study, Pennington founded the Philadelphia Clinical Laboratory, where she performed bacteriological analyses for physicians. Soon she became director of the bacteriological laboratory of the Philadelphia Department of Health. There she developed techniques and standards for milk inspection and dairy product preservation that came into use throughout the country.

In 1907 Pennington joined the staff of the Bureau of Chemistry of the United States Department of Agriculture after taking the civil service examination as "M. E. Pennington" so it would not be apparent that she was a woman. In the following year, Pennington was named director of the Food Research Laboratory, a new division within the Bureau of Chemistry. She continued working out of her own lab in Philadelphia, where she investigated and devised methods of storing and shipping poultry, eggs, and other perishable foods without spoilage. She established national standards for ice-cooled refrigerator cars and solved the problem of humidity control in cold storage.

In 1922 Pennington became a private consultant in New York City. During the next thirty years, she contributed to advances in the design and construction of home and commercial refrigeration units and conducted research that revealed the best ways to freeze many different foods. Pennington's life work contributed greatly to the successful storage and transportation of perishable foods of all varieties.