Calpain‐2 levels are higher in colorectal tumors resistant to chemotherapy and previous work showed calpain‐2 inhibitor therapy reduced inflammation‐driven colorectal cancer, but direct effects of the inhibitor on colon cancer cells themselves were not demonstrated. In the present study, five human colon cancer cell lines were directly treated with a calpain‐2 inhibitor and results showed increased cell death in 4 of 5 cell lines and decreased anchorage‐independent growth for all cell five lines. When tested for levels of calpain‐2, three cell lines exhibited increasing levels of this enzyme: HCT15 (low), HCC2998 (medium), and HCT116 (significantly higher). This was consistent with gel shift assays showing that calpain‐2 inhibitor reduced of NF‐κB nuclear translocation most effectively in HCT116 cells. Ability of calpain‐2 inhibitor to impede tumor progression in vivo was evaluated using intrarectal transplant of luciferase‐expressing cells for these three cell lines. Results showed that calpain‐2 inhibitor therapy reduced tumor growth and increased survival only in mice injected with HCT116 cells. These data suggest calpain‐2 inhibitor treatment may be most effective on colorectal tumors expressing highest levels of calpain‐2.