Decidualization of endometrial stromal cells (ESCs) is critical for embryo implantation and maintenance of pregnancy. Proprotein convertase (PC) 5/6 is suggested to play an important role in the processes of stromal cell decidualization and embryo implantation in the mouse. PC5/6 is a member of the PC family responsible for processing precursor proteins to their active forms by selective proteolysis. In this study, we investigated the regulation of PC5/6 mRNA and protein expression in human ESCs during decidualization in vitro. Real-time PCR analyses revealed a significant increase in PC5/6 mRNA levels in ESCs treated with 17β-estradiol (E2) plus medroxyprogesterone acetate during decidualization. On the other hand, E2 alone did not increase PC5/6 mRNA expression. Intense PC5/6 immunoreactivity was observed in the cytoplasm of E2 plus medroxy-progesterone acetate-treated ESCs (decidualized ESCs) compared with E2-treated ESCs on d 12 of culture (nondecidualized ESCs). This PC5/6 immunoreactivity was abolished by cotreatment with ZK 98299, a progesterone receptor antagonist. Western blotting revealed PC5/6 as approximately 120-kDa bands (pro- and mature forms) and a 65-kDa band (C-terminally truncated form) in decidualized ESCs. Using an antisense morpholino approach, prolactin production, a typical marker for decidualization, was significantly attenuated in decidualized ESCs after treatment with PC5/6 morpholino antisense oligonucleotides in comparison with controls. These results suggest that PC5/6 plays a key role for decidualization in human endometrium.