Corticotropin-releasing hormone (CRH) and connexin 43 (Cx43) play crucial roles in uterine contraction and the onset of labor. The aim of the present study was to investigate the regulatory effects of CRH on Cx43 expression in human myometrial smooth muscle cells (SMCs) and, potentially, its activation of the c-Fos/activator protein (AP)-1 signaling pathway. Human myometrial SMCs collected from nonpregnant women were treated with different concentrations of CRH. Transient transfection of AP-1 decoy oligodeoxynucleotide (ODN) was used to block AP-1 sites of Cx43. The transcriptional activity of AP-1 was detected by luciferase assay. Cx43 protein expression was visualized by immunofluorescence staining. mRNA and protein expression of c-Fos and Cx43 were demonstrated by real-time quantitative RT-PCR and Western blot, respectively. CRH facilitated Cx43 expression and enhanced AP-1 promoter activity in human uterine SMCs. After CRH treatment, Cx43 expression in the cytoplasm increased significantly. CRH significantly increased mRNA and protein expression of c-Fos and Cx43 in a dose-dependent manner (P < 0.01). A transient transfection of AP-1 decoy ODN did not affect CRH regulation of c-Fos (P > 0.05) but almost completely abolished CRH-induced enhancement of Cx43 expression (P < 0.01). In human primary myometrial SMCs, CRH enhances Cx43 mRNA and protein expression through upregulation of c-Fos expression. Blockade of AP-1 sites to the Cx43 promoter can neutralize the CRH-induced upregulation of Cx43.