Anticancer activity of diospyrin and its derivatives (1–5) was evaluated against thirteen human cell lines. Compared to diospyrin (1), the acetylamine derivative (4) exhibited increase in cytotoxicity, particularly in HT-29 colon cancer cells, showing GI50 values of 33.90 and 1.96 μM, respectively. Also, enhanced toxicity was observed when cells, pre-treated with compound 4, were exposed to radiation. In vivo assessment of 4 was undertaken on tumour-bearing Nod-Scid mice treated at 4mg/kg/day. Significant reduction in relative tumour volume (~86–91 %) was observed during the 12th–37th days after drug treatment. Increased caspase-3 activity and DNA ladder formation was observed in HT-29 cells after treatment with 4, suggesting induction of apoptotic death after drug treatment. Moreover, flow cytometric determination of Annexin V- FITC positive and PI negative cells demonstrated 17.4, 26.4, and 27.9 % of early apoptosis, respectively, upon treatment with 5, 10 and 25 μM of 4. HT-29 cells after treatment with 4 (1–25 μM) revealed ~2.5- 3- folds generation of ROS. Furthermore, concentration dependent decrease of mitochondrial trans-membrane potential (Δψm), and expression of Bcl-2/Bax and other marker proteins suggested involvement of mitochondrial pathway of cell death. Overall, our results demonstrated the underlying cell-death mechanism of the plant-derived naphthoquinonoid (4), and established it as a prospective chemotherapeutic ‘lead’ molecule against colon cancer.