Most Gram-positive bacteria are susceptible to the bactericidal action of fatty acids and bile acids. Because dietary calcium phosphate (CaP(i)) lowers the intestinal concentration of these antimicrobial agents, high CaP(i) intake may enhance intestinal colonization of Gram-positive pathogens and the subsequent pathogenesis. In this study, we tested this hypothesis in a rat model using Listeria monocytogenes. Rats were fed diets containing low (20 micromol/g diet) or high (160 micromol/g diet) amounts of CaP(i). Dietary fat was provided as corn oil or milk fat. Rats were orally inoculated with L. monocytogenes. When rats consumed diets containing corn oil, high CaP(i) intake indeed stimulated colonization of L. monocytogenes and increased L. monocytogenes translocation and diarrhea. In addition, supplemental CaP(i) enhanced ex vivo growth of L. monocytogenes in fecal extracts of rats fed corn oil diets, suggesting that high CaP(i) intake decreased a luminal inhibitory factor. The concentrations of bile salts and fatty acids, which were highly listericidal in vitro, were indeed considerably decreased in fecal water of rats in the high calcium corn oil group. Surprisingly, dietary CaP(i) did not affect colonization and translocation of L. monocytogenes in rats fed the milk fat diet, nor did CaP(i) enhance ex vivo growth in fecal extracts. This absence of Listeria stimulation was associated with a lack of effect of dietary CaP(i) on fecal soluble fatty acids. In addition, residual soluble bile salts were higher in rats fed the high CaP(i) milk fat diet compared with the high CaP(i) corn oil diet. These results suggest that the stimulating effect of CaP(i) on L. monocytogenes infection depends on the type of dietary fat consumed.