To evaluate the in vitro anticancer activity and to investigate the mechanism of action of a thiophene heterocyclic compound, [3-Amino-5-[(2,6-dimethylphenyl)amino]-4-(phenylsulfonyl)-2-thienyl](4-fluorophenyl)methanone (APTM) against human colon cancer HCT116 cells. Sulforhodamine B assay and colony formation assay for cell proliferation assay; propidium iodide (PI) staining for cell cycle profile analysis; Hoechst staining; annexin V-FITC/PI double staining and Western blotting for apoptosis assay. APTM inhibits the growth of HCT116 cells dose and time dependently. The growth inhibitory effect of APTM on HCT116 cells was associated with induction of apoptosis but not cell cycle arrest. Also, the isogenic cell depletion of p53 was resistant to APTM-induced apoptosis and thus grows relatively better than the wild-type cells. The anticancer effect of APTM resulted from p53-dependent induction of apoptosis. Also, APTM is a promising lead compound for the treatment of human colon cancer.