Prolyl oligopeptidase (POP), one of the most widely distributed serine endopeptidases, is highly expressed in the ovaries. However, the physiological role of POP in the ovaries is not clear. In this study, we investigated the significance of POP in the corpus luteum. Murine luteal cells were cultured in vitro and treated with a POP selective inhibitor, (2S)‐1[[(2
S)‐1‐(1‐oxo‐4‐phenylbutyl)‐2‐pyrrolidinyl carbonyl]‐2‐pyrrolidinecarbonitrile (KYP‐2047). We found that KYP‐2047 treatment decreased progesterone secretion. In contrast, POP overexpression increased progesterone secretion. Three essential steroidogenic enzymes, including p450 cholesterol side‐chain cleavage enzyme (CYP11A), 3β‐hydroxysteroid dehydrogenase (3β‐HSD), and the steroidogenic acute regulatory protein (StAR), were regulated by POP. Further studies showed that POP overexpression increased ERK1/2 phosphorylation and increased the expression of steroidogenic factor 1 (SF1), while KYP‐2047 treatment decreased ERK1/2 phosphorylation and SF1 expression. To clarify the role of ERK1/2 signaling in POP‐regulated progesterone synthesis, U0126‐EtOH, an inhibitor of the ERK signaling pathway, was used to treat luteal cells. We found that U0126‐EtOH decreased progesterone production and the expression of steroidogenic enzymes and SF1. POP overexpression did not reverse the effects of U0126‐EtOH. Overall, POP regulates progesterone secretion by stimulating the expression of CYP11A, 3β‐HSD, and StAR in luteal cells. ERK signaling and downstream SF1 expression contribute to this process.