Urocortins (UCNs), belonging to corticotropin-releasing hormone (CRH) family, exert their function via CRH receptor type 1(CRHR1) and 2 (CRHR2). Our previous studies have demonstrated that CRH acts on CRHR1 to potentiate prostaglandin (PG) output induced by inflammatory stimuli in myometrial cells. In the present study, we sought to investigate the effects of UCNs on prostaglandin (PG) output via CRHR2 in cultured human uterine smooth muscle cells (HUSMCs) from human term myometrium. We found that UCN and UCN3 treatment promoted PGE2 and PGF2α secretion in a dose-dependent manner. In contrast, UCN2 dose-dependently inhibited PGE2 and PGF2α secretion. Their effects could be reversed by CRHR2 antagonist and CRHR2 siRNA. Mechanically, we showed that UCN and UCN3 suppressed cAMP production and led to Gi activation, while UCN2 promoted cAMP production and activated Gs signaling. Further, UCN and UCN3 could activate NF-κB and MAPK signaling pathways. These effects were dependent on Gi signaling. In contrast, UCN did not activate MAPK and NF-κB signaling. UCN and UCN3 stimulation of PG secretion was dependent on Gi/adenylyl cyclase (AC)/cAMP, NF-κB and MAPK signaling pathways, while UCN2 suppression of PG output was through Gs/AC/cAMP signaling pathways. Our data suggest that UCN, UCN2 and UCN3 can finely regulate the secretion of PGs via CRHR2, which facilities the functional status of uterus during pregnancy.