BACKGROUND Silver nanoparticles (AgNP) are commonly used metallic nanoparticles in health care systems. Colon cancer incidence is increasing worldwide. In this study, AgNP was synthesized using β-sitosterol and its cytotoxic potential was evaluated in human colon cancer (HT-29) cells. METHODS Characterization of AgNP was analyzed by TEM and spectrophotometry analysis. HT-29 cells were treated with different concentrations (2, 4, 6, 8 and 10 ng/ml) of AgNPs and cytotoxicity was evaluated by MTT assay. The apoptosis was analyzed by the flow cytometry. The expression of p53 protein was analyzed by western blotting. RESULTS β-sitosterol mediated AgNP are spherical in shape and induced concentration-dependent cytotoxicity in HT-29 cells. AgNP caused apoptosis related morphological changes as evidenced by annexin positive staining. AgNP treatments also induced the p53 expression in HT-29 cells. CONCLUSIONS Our present result suggests that β-sitosterol mediated AgNP induce apoptosis in colon cancer cells and this finding may pave the way for further experimental analysis in vivo.