A novel strategy using cryo-shocked macrophages to deliver attenuated Salmonella typhimurium (VNP20009) for tumor-targeted therapy has been developed. This approach involves loading macrophages with VNP20009 and then subjecting them to liquid nitrogen cold shock to create "dead" yet "functional" cells that can deliver the bacteria to tumors while minimizing toxicity. The cryo-shocked macrophages retain their structural integrity and tumor-targeting ability, allowing the bacteria to be released and proliferate within the tumor microenvironment. This strategy effectively avoids immune activation and neutrophil-mediated clearance of the bacteria, enhancing their tumor enrichment and antitumor efficacy. The macrophages also help activate the tumor microenvironment by increasing the number of antitumor effector cells (such as M1-like macrophages and CD8+ T cells) and reducing the number of protumor effector cells (such as M2-like macrophages and CD4+ Tregs). The cryo-shocked macrophage-mediated delivery of VNP20009 significantly improves antitumor efficacy in a subcutaneous H22 tumor model. This strategy offers a promising approach for the clinical translation of live bacterial therapies for cancer, as it enhances biosafety and tumor targeting while maintaining the therapeutic activity of the bacteria. The cryo-shocked macrophages can be easily prepared, stored, and used for targeted delivery, making them a valuable tool for bacterial-based cancer therapy. The results suggest that this strategy could be further optimized for clinical applications by modifying the macrophages and bacteria to enhance their tumor-targeting and therapeutic effects.A novel strategy using cryo-shocked macrophages to deliver attenuated Salmonella typhimurium (VNP20009) for tumor-targeted therapy has been developed. This approach involves loading macrophages with VNP20009 and then subjecting them to liquid nitrogen cold shock to create "dead" yet "functional" cells that can deliver the bacteria to tumors while minimizing toxicity. The cryo-shocked macrophages retain their structural integrity and tumor-targeting ability, allowing the bacteria to be released and proliferate within the tumor microenvironment. This strategy effectively avoids immune activation and neutrophil-mediated clearance of the bacteria, enhancing their tumor enrichment and antitumor efficacy. The macrophages also help activate the tumor microenvironment by increasing the number of antitumor effector cells (such as M1-like macrophages and CD8+ T cells) and reducing the number of protumor effector cells (such as M2-like macrophages and CD4+ Tregs). The cryo-shocked macrophage-mediated delivery of VNP20009 significantly improves antitumor efficacy in a subcutaneous H22 tumor model. This strategy offers a promising approach for the clinical translation of live bacterial therapies for cancer, as it enhances biosafety and tumor targeting while maintaining the therapeutic activity of the bacteria. The cryo-shocked macrophages can be easily prepared, stored, and used for targeted delivery, making them a valuable tool for bacterial-based cancer therapy. The results suggest that this strategy could be further optimized for clinical applications by modifying the macrophages and bacteria to enhance their tumor-targeting and therapeutic effects.