Calpains are a family of calcium-dependent proteases, which modulate their substrates rather than degrade them in such a way that modifies them. Calpains deregulations have been determined as an aggravating factor of different diseases, including cancer. Nevertheless, there are no clear records about the particular contribution of each calpain isoform in physiological processes and how these isoforms are deregulated in pathological conditions. In vivo, each calpain isoform recognizes specific proteins as substrates, and it has been suggested that calpains subcellular localization might determine their substrate recognition, and consequently their functions. In the present study we have explored the relationship between calpain-1 and calpain-2 isoforms subcellular localization with their involvement in different cell functions, such as cell adhesions cleavage and cell differentiation. Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. To evaluate the isoform-specific CAPN involved in the cleavage of adhesion complexes we used murine mammary gland during involution and breast cancer cell lines as physiological and pathological study models, respectively. We observed a switch in the calpain isoform responsible of this cleavage depending on the biological context: during mammary gland involution, calpain-2 was the isoform ruling this function, while in breast cancer cell lines was calpain-1. In addition, during the second phase of murine mammary gland involution, calpain-1 was gathered in the nuclear compartment of differentiating adipocytes. The relationship between localization and isoform-specific functions of each calpain isoform were explored in the differentiation model of 3T2-L1 cell line. Both isoforms showed a dynamic expression and localization changes along the differentiation course from pre-adipocytes to adipocytes, changes that determined their functions in the differentiation process.