In breast cancer, progression from localized stage I to distant metastatic stage IV disease is associated with a reduction of 5-year survival from nearly 100% to 23.2%. Expression of the calcium-activated protease isoforms calpain-1 and calpain-2 has been correlated with cell migration and invasion in vitro, metastatic potential in preclinical mouse models of cancer, and breast cancer prognosis in patients. It is unclear which of these two calpain isoforms is responsible for the apparent metastatic potential of cancer cells. Here, we demonstrate that while individual CRISPR-Cas9 knockouts of either CAPN1 or CAPN2 genes (encoding the catalytic subunits of calpain-1 and -2, respectively) reduce in vitro migration and marginally suppress in vivo metastasis, genetic disruption of both calpain-1 and calpain-2 through knockout of the CAPNS1 gene (encoding the common regulatory subunit of calpain-1 and -2) diminishes metastasis by 83.4 ± 13.6% in a mouse xenograft model of human triple-negative breast cancer. The effect of calpain-1/2 deficiency was replicated in vitro with a modified cell-permeable calpastatin (CAST)-based peptide inhibitor (cell migration reduced to 53.5 ± 11.0% of vehicle control). However, this peptide inhibitor was not effective in vivo at reducing metastasis under the conditions used (vehicle vs. CAST, 1.12 ± 1.35 lung metastases per mm² vs. 0.34 ± 0.20 metastases per mm²), likely due to rapid clearance, as indicated by the short serum half-life. This work demonstrates that calpain-1/2 disruption effectively abrogates metastasis and provides rationale for development of effective calpain inhibitors.