Anno was born in 1926 in Japan, and grew up in the small town of Tsuwano. As a student at a regional high school, he studied art, drawing, and the writings of Hermann Hesse. During World War II, Anno was drafted into the Japanese army. After the war, Anno earned a degree from the Yamaguchi Teacher Training College in 1948. He taught mathematics for ten years in an elementary school in Tokyo before beginning a career illustrating children's books. Anno lives in Japan with his wife, Midori. They have two children, Masaichiro and Seiko.

Anno is best known for wordless picture books featuring small, detailed figures. In the "Journey" books, a tiny character travels through a nation's landscape, densely populated with pictures referencing that country's art, literature, culture, and history. Anno's illustrations are often in pen and ink and watercolor, and occasionally incorporate collage and woodcuts. They are intricately detailed, showing a sense of humor as well as an interest in science, mathematics, and foreign cultures. They frequently incorporate subtle jokes and references. Anno's style has been compared to that of M. C. Escher. Although he is best known for his children's books, his paintings have earned recognition in his native Japan. In Tsuwano, Japan, the Mitsumasa Anno Museum houses a collection of his works.