Background: Colorectal cancers global incidence is increasing due to the rapid acquisition of Western lifestyle habits such as high caloric nutrition and sedentary status. Natural bioactive products play critical roles in anticancer drug development. Aim: The aim of the study was to investigate the antiproliferative and proapoptotic effects of vanillin (VL) an active constituent from Vanilla bean in colon cancer (human colorectal adenocarcinoma cell line [HT-29]) cells. Methodology: We demonstrated the cell growth and apoptosis in HT-29 cells. The apoptotic measures were analyzed by analyzing mitochondrial membrane potential (Δψm) level; apoptotic bodies by acridine orange/ethidium bromide and Hoechst staining. Results: Our results indicated that VL induces apoptosis as evidenced by cell viability loss, resulting in the altered Δψm in HT-29 cells. Further, the prooxidant role of VL inhibit B-cell lymphoma 2 (Bcl2) expression with the simultaneous upregulation of BCL-2 associated X, Cytochrome c, Capspase-9 and 3 protein expressions in HT-29 cells. Conclusion: Thus, in this regard, we have concluded that VL induces apoptosis in HT-29 cells through inducing oxidative damage and modulates apoptotic marker expressions. VL may therefore be used as a therapeutic agent for colon cancer treatment.