Gossypol is a polyphenolic, yellowish compound derived from cottonseed extract. The present study examined the effects of gossypol on the apoptosis and autophagy of HT‑29 cells. A Cell Counting Kit‑8 assay, Annexin V‑FITC, JC‑1 staining and western blotting were used to identify the viability of cells, stages of apoptosis and the expression levels of the signaling proteins. Gossypol promoted apoptosis and induced the loss of mitochondrial membrane potential. Further investigation of the apoptotic mechanism revealed that gossypol increased the ratio of B‑cell lymphoma 2 (Bcl-2)-associated X protein/Bcl‑2 protein levels and upregulated the expression of caspase‑3. Gossypol also enhanced the activity of microtubule‑associated protein light chain 3 LC3‑II and Beclin‑1 and downregulated LC3‑I, in a dose‑dependent manner. Together, these finding suggested that gossypol may be a novel and potential antitumor agent.