Abstract Honeybee (Apis melifera) venom (HBV), which includes melittin and lipid-soluble ingredients (chrysin and pinocembrin), elicited increases in the CD4+/CD8+ T lymphocyte ratio, relative mRNA expression levels of the T helper type 1 (Th 1) cytokines (interferon-γ and IL-12) and reinforced viral clearance of an experimental porcine reproductive and respiratory syndrome (PRRS) virus infection in our previous study. On the basis of that previous study, we have now developed poly-d,l-lactide-co-glycolide (PLGA)-encapsulated HBV nanoparticles (P-HBV) for longer sustained release of HBV. We administered P-HBV to pigs via the rectal route, and then evaluated the potential immune-enhancing and bacterial clearance effects of P-HBV against Salmonella enterica serovar Typhimurium. The CD4+/CD8+ lymphocyte ratio, proliferative capacity of peripheral blood lymphocytes and relative mRNA expression levels of IFN-γ and IL-12 (produced mainly by Th1 lymphocytes) were significantly increased in the P-HBV group up to 2 weeks post-administration of P-HBV. After S. Typhimurium infection, the P-HBV group showed a marked reduction in microbial burden in feces and all tissue samples (including the ileum, cecum, colon, and mesenteric lymph node (MLN)), a significant increase in Th 1 cytokines (IFN-γ, IL-2, and IL-12) and a marked decrease in a Th 2 cytokine (IL-4) in all tissue samples and peripheral blood lymphocytes. Thus, P-HBV may be a promising strategy for immune enhancement and prevention of S. Typhimurium or other bacterial infections.