In this review, we have tried to summarize the evidence and molecular characterization indicating that 20α-hydroxysteroid dehydrogenase (20α-HSD) is a group of the aldo-keto reductase (AKR) family, and it plays roles in the modulation and regulation of steroid hormones. This enzyme plays a critical role in the regulation of luteal function in female mammals. We have studied the molecular expression and regulation of 20α-HSD in cows, pigs, deer, and monkeys. The specific antibody against bovine 20α-HSD was generated in a rabbit immunized with the purified recombinant protein. The mRNA expression levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. The mRNA was also specifically detected in the placental and ovarian tissues during pregnancy. The 20α-HSD protein was intensively localized in the large luteal cells and placental cytotrophoblast villus, glandular epithelial cells of the endometrium, syncytiotrophoblast of the placenta, the isthmus cells of the oviduct, and the basal part of the primary chorionic villi and chorionic stem villus of the placenta and large luteal cells of the CL in many mammalian species. Further studies are needed to determine the functional significance of the 20α- HSD molecule during ovulation, pregnancy, and parturition. This article will review how fundamental information of these enzymes can be exploited for a better understanding of the reproductive organs during ovulation and pregnancy.