Objective: Corticotrophin-releasing hormone (CRH) and Connexin 43 (Cx43) play crucial roles in uterine contraction and the onset of labor. The aim of this study is to investigate the regulatory effects of CRH on Cx43 expression in human myometrial cells and its potential signaling pathway of c-Fos/activator protein-1 (AP-1) activation. Methods: Human myometrial cells collected from nonpregnant women were treated with CRH at different concentration. A 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 immunoflorescence staining. mRNA and protein expression of c-Fos and Cx43 were demonstrated by real-time RT-PCR quantitation and Western blot, respectively. Results: CRH facilitated Cx43 expression and enhanced AP-1 promoter activity in human uterine smooth muscle cells. After being treated with CRH, the expression of Cx43 in the cytoplasm of the cells increased significantly. CRH significantly increased mRNA and protein expression of c-Fos and Cx43 in a dose-dependent manner (p<0.01, respectively). A transient transfection of AP-1 decoy ODN did not affect the regulation of CRH on c-Fos (p>0.05). However, AP-1 decoy ODN almost completely abolished the enhancement of CRH on Cx43 expression (p<0.01). Conclusions: In human primary myometrial smooth muscle cells, CRH enhances Cx43 mRNA and protein expression through an up-regulation of c-Fos expression. A blocking of AP-1 sites to Cx43 promoter can neutralize the up-regulation effects of CRH on Cx43.