The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes progesterone into a biologically inactive steroid, 20α-dihydroprogesterone (20α-OHP). In the corpora lutea of rats and mice, 20α-HSD is considered to be involved in functional luteolysis. It is also distributed in other tissues including the placenta, endometrial epithelia and fetal skin, although the roles it plays in these tissues remain to be elucidated. In the present study, we investigated the role of 20α-HSD in the maintenance of pregnancy using mice with targeted disruption of the 20α-HSD gene. We first confirmed that the number of pups was significantly smaller in 20α-HSD^-/- pairs than in 20α-HSD ^+/+ pairs. We then mated 20α-HSD^+/- males and females so that each pregnant female produced 20α-HSD^+/+, 20α-HSD^+/- and 20α-HSD^-/- offspring. The genotype ratio of the offspring did not match the Mendel's law of inheritance, and the numbers of 20α-HSD^+/- and 20α-HSD^-/- offspring were smaller than expected values. Although the genotype ratio of fetuses on days 13, 15 and 18 of pregnancy matched the Mendel's law, the total number of fetuses on day 18 was significantly smaller than that on day 13, suggesting that fetal loss occurred during late pregnancy. Next, we transferred 20α-HSD^+/+ embryos to 20α-HSD^+/+ or 20α-HSD^-/- females and found that the number of offspring was significantly smaller in 20α-HSD^-/- dams than in 20α-HSD^+/+ dams. Expression of 20α-HSD mRNA in the fetus, placenta and uterus progressively increased from day 11 to 18 of pregnancy. In addition, concentrations of progesterone were significantly higher in the 20α-HSD^-/- fetuses than in the 20α-HSD^+/+ fetuses, while those of 20α-OHP were lower in the 20α-HSD^-/- fetuses than in the 20α-HSD^+/+ fetuses. These results suggest that both maternal and fetal 20α-HSD play a role in maintaining normal pregnancy at least partially by reducing progesterone concentrations in fetuses.