Attenuated Salmonella typhimurium VNP20009 is a novel oncolytic bacterium with high tumor-targeting properties. One of its anti-tumor mechanisms is the induction of tumor cell apoptosis, although the specific molecular mechanisms remain unclear. Melanoma, the deadliest form of skin cancer, is associated with significant challenges, such as severe side effects and high recurrence rates in current treatments. This study used the B16F10 mouse melanoma cell line as a model to explore the regulatory mechanism of VNP20009-induced apoptosis in melanoma cells. The results showed that VNP20009 significantly induced apoptosis in B16F10 cells in a time- and concentration-dependent manner. Transcriptomic analysis revealed that the p53 signaling pathway was significantly enriched in the VNP20009-treated group, suggesting that this pathway might mediate the pro-apoptotic effects of VNP20009. Further investigations demonstrated that VNP20009 induces apoptosis by activating key genes in the p53 pathway, including PUMA, and its upstream and downstream molecules, such as p53, CytC, CASP9, and CASP3, forming a cascade reaction. In conclusion, this study elucidates the molecular mechanism by which VNP20009 induces apoptosis in B16F10 melanoma cells through the p53-PUMA axis, providing new theoretical insights for melanoma treatment based on attenuated Salmonella bacteria.