INTRODUCTION: Concerns about antibiotic resistance have prompted interest in alternative strategies for enhancing disease resistance, particularly in livestock and poultry production. OBJECTIVES: This study explored the role of trained immunity in enhancing resistance to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection in mice and chickens. METHODS: We investigated the effects of probiotics and inactivated pathogenic bacterial strains on host immunity in Toll-like receptor 2-deficient mice (TLR2 RESULTS: Probiotics and inactivated pathogenic strains enhanced host immunity and protected against S. Typhimurium infection. As demonstrated in the TLR2-deficient mice, the effects were not dependent on bacterial outer membrane components. Instead, bacterial genomes played a significant role in activating trained immunity. Macrophages were identified as the primary cells that mediated the response with the cGAS-STING pathway playing a crucial role. The results observed using the mouse models led to investigating the potential application of trained immunity in poultry. CONCLUSION: Trained immunity activated by probiotics and inactivated bacterial pathogens enhanced resistance against S. Typhimurium infection via macrophage activation and involved the cGAS-STING pathway. These findings highlight the potential of trained immunity as an alternative strategy for disease prevention in both livestock and poultry.