Pristimerin is a naturally occurring triterpenoid that has a cytotoxic effect on several cancer cell lines. However, the cytotoxic effects of pristimerin as well as its molecular mechanisms of action against colorectal cancer have never been explored. In the present study, we investigated the anticancer potential of pristimerin, and examined the different signaling pathways affected by its action in three colon cancer cell lines namely HCT-116, COLO-205 and SW-620. Pristimerin was found to possess potent cytotoxic and proliferation inhibitory effects against these cell lines. Cell cycle analysis revealed G1 phase arrest, which was strongly associated with decreased expression of cyclin D1 and cyclin-dependent kinases (cdk4 and cdk6) with concomitant induction of p21. Pristimerin also induced apoptosis in a dose-dependent manner. Cell plasma membrane alterations studied by Annexin V/PI double staining, loss of mitochondrial membrane potential (ΔΨm), measurements of caspase activities and the inhibitory effect of Z-VAD-FMK (a caspase inhibitor) confirmed the apoptotic effect of pristimerin. Moreover, western blot data showed that apoptotic induction was associated with activated caspase-3 and -8, PARP-1 cleavage and modulation of the expression levels of Bcl-2 family proteins. Additionally, pristimerin treatment downregulated the phosphorylated forms of EGFR and HER2 proteins, and subsequently caused a decrease in the phosphorylated forms of Erk1/2, Akt, mTOR and NF-κB proteins. Taken together, these results suggest that pristimerin may have potential as a new targeting therapeutic strategy for the treatment of colon cancer.