The calpain family of proteases has been implicated in cellular processes such as apoptosis, proliferation and cell migration. Calpains are involved in several key aspects of migration, including: adhesion and spreading; detachment of the rear; integrin- and growth-factor-mediated signaling; and membrane protrusion. Our understanding of how calpains are activated and regulated during cell migration has increased as studies have identified roles for calcium and phospholipid binding, autolysis, phosphorylation and inhibition by calpastatin in the modulation of calpain activity. Knockout and knockdown approaches have also contributed significantly to our knowledge of calpain biology, particularly with respect to the specific functions of different calpain isoforms. The mechanisms by which calpain-mediated proteolysis of individual substrates contributes to cell motility have begun to be addressed, and these efforts have revealed roles for proteolysis of specific substrates in integrin activation, adhesion complex turnover and membrane protrusion dynamics. Understanding these mechanisms should provide avenues for novel therapeutic strategies to treat pathological processes such as tumor metastasis and chronic inflammatory disease.