In the present study, we demonstrated that repeated treatment with recombinant human prolactin (rhPRL) protected mice against Salmonella typhimurium infection. The protective activity was statistically significant, dose-dependent and present only when rhPRL treatments were performed before the infection. This activity was probably related to the observed increases in phagocytosis and intracellular killing of peritoneal macrophages induced by the hormonal treatment. The number of peripheral leukocytes was not modified, excluding a mobilization of cells from other compartments. A decrease in the mortality rate after challenge was also observed in mice treated with the monoclonal antibody anti-PRL receptor U5, confirming that the protective activity was associated with receptor activation. Our studies also suggest that nitric oxide (NO) production was involved in the protective effect of rhPRL since pre-treatment of the animals with L-NAME, an inhibitor of NO-synthase, was able to completely revert the protective activity, whereas D-NAME, the inactive D-isomer, was without effect.