In 2000 she was selected for her second flight as part of the crew of STS-107. This mission was repeatedly delayed due to scheduling conflicts and technical problems such as the July 2002 discovery of cracks in the shuttle engine flow liners. On January 16, 2003, Chawla finally returned to space aboard Columbia on the ill-fated STS-107 mission. Chawla was dedicated to the scientific goals of spacehab or freestar microgravity research mission, for which the crew conducted nearly 80 experiments studying earth and space science, advance technology development, and astronaut health and safety.

Chawla died in the Space Shuttle Columbia disaster which occurred on February 1, 2003, when the Space Shuttle disintegrated over Texas during re-entry into the Earth's atmosphere, with the loss of all seven crew members, shortly before it was scheduled to conclude its 28th mission, STS-107. US space shuttle Columbia disintegrated shortly before landing on Saturday morning, killing the seven crew members on board, including Indian-American Kalpana Chawla. She died a hero and a role-model for many young women, particularly those in her hometown of Karnal where she periodically returned to encourage young girls to follow in her footsteps.