The present study was to investigate the anticancer effect of chloroquine on proliferation of mouse colon cancer cell line CT26 in vivo and in vitro and the possible mechanism. We found that chloroquine inhibited CT26 proliferation by concentration- and time-dependent manner. This effect was associated with apoptosis induction and decreased level of phosphorylated p42/44 mitogen-activated protein kinase and phosphorylated Akt. The in vivo study showed chloroquine-reduced tumor volume and prolonged survival time in CT26-bearing mice. These observations indicated chloroquine could inhibit CT26 proliferation by inducing apoptosis both in vitro and in vivo, providing its chemotherapeutic potential of human cancers.