ObjectiveTwo serine protease inhibitors, peptidase inhibitor 3 (PI3) and secretory leukocyte protease inhibitor (SLPI), play important roles in protease inhibition and antimicrobial activity, but their expression, regulation, and function at the maternal–fetal interface in pigs are not fully understood. Therefore, we determined the expression and regulation of PI3 and SLPI in the endometrium throughout the estrous cycle and at the maternal–fetal interface in pigs.MethodsEndometrial tissues during the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid to late pregnancy were obtained, and the expression ofPI3andSLPIwas analyzed. The effects of the steroid hormones estradiol-17β (E2) and progesterone (P4) on the expression ofPI3andSLPIwere determined in endometrial explant cultures.ResultsPI3andSLPIwere expressed in the endometrium during the estrous cycle and pregnancy, with higher levels during mid to late pregnancy than during the estrous cycle and early pregnancy. Early-stage conceptuses and chorioallantoic tissues during mid to late pregnancy also expressedPI3andSLPI. PI3 protein andSLPImRNA were primarily localized to endometrial epithelia. In endometrial explant cultures, the expression ofPI3was induced by increasing doses of P4, and the expression ofSLPIwas induced by increasing doses of E2 and P4.ConclusionThese results suggest that thePI3andSLPIexpressed in the endometrium and conceptus tissues play an important role in antimicrobial activity for fetal protection against potential pathogens and in blocking protease actions to allow epitheliochorial placenta formation.