Background and aim: One of the active combustion product of cigarette smoke, Benzo[a]pyrenes, role in pulmonary cancer is clearly understood. However, its role in gastrointestinal cancer including colon cancer is not clearly understood. Methods: In this study, benzo(a)pyrenes was treated to colon cells to evaluate its role in cell viability, cellular ROS, and gene expression of various PLA2 isoforms was evaluated by FACS and PCR. The identified PLA2 was silenced at the gene level to evaluate its role in cell viability and ROS generation. Results: B(a)P treatment at 1 µg/mL for 48 h to HCT-15 male colon cells significantly reduced the cell viability without affecting HT-29 female colon cells. Higher doses and longer treatment duration with B(a)P showed that female colon cells were highly sensitive than male colon cells. Annexin-V/PI staining for preapoptotic detection showed that B(a)P treatment increased the apoptosis in both the cell types in a concentration and time-dependent manner. The cytosolic ROS (cROS) and superoxide radical (SOR) formation in the female colon cells was significantly higher than male colon cells unlike the mitochondrial ROS (mtROS) production which was significantly higher in male colon cells. Treatment with B(a)P significantly upregulated the IID and IVA PLA2 isoform groups in HCT-15 male colon cells, whereas IB was upregulated in HT-29 female colon cells among the various PLA2 isozyme gene studied (IB, IID, III, IVA, IVB, IVC, VI, X, aiPLA2 and iPLA2). Gene silencing experiments targeting PLA2 IID and IVA in the HCT-15 male colon cells and IB in HT-29 female colon cells showed no effect with B(a)P treatment on the cell proliferation, apoptosis, membrane integrity and free radicals (ROS, mtROS, and SOR) generation. Conclusion: Targeting specific PLA2 isozymes in a cell-specific manner abolished the B(a)P-induced PLA2-mediated oxidative damage–related signaling pathways.