We previously demonstrated that progesterone (P4) up-regulates p53 expression in human umbilical venous endothelial cells (HUVECs) through P4 receptor (PR) activation of extranuclear signaling pathways. However, the involvement of nuclear PR in P4-increased p53 expression is still unclear. Here, the molecular mechanism underlying PR-regulated p53 expression in HUVECs was investigated. Treatment with P4 increased nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha phosphorylation (IkappaBalpha and nuclear factor-kappaB (NFkappaB) nuclear translocation. Interestingly, P4 also increased PR-A, but not PR-B, nuclear translocation in HUVECs. Immunoprecipitation assay illustrated that P4 increased the formation of PR-A-NFkappaB complex in both the cytosol and the nucleus of HUVEC. Chromatin immunoprecipitation assay showed an interaction between PR and the NFkappaB binding motif on the p53 promoter. Ablation of the NFkappaB binding motif in the p53 promoter completely abolished P4-increased p53 promoter activity. In the absence of P4, overexpression of NFkappaB did not increase NFkappaB nuclear translocation. In contrast, treatment of NFkappaB-overexpressing HUVECs with P4 for only 4 hours, which is much shorter than the time (21.5 h) required for P4-induced IkappaBalpha phosphorylation, increased NFkappaB nuclear translocation. Blockade of PR activity abolished this effect. Taken together, these results uncover a novel role of PR for P4-induced NFkappaB nuclear translocation and suggest that PR-A-NFkappaB complex formation is required for NFkappaB nuclear translocation and binding onto the p53 promoter in HUVECs. Our data indicate that both nuclear and extranuclear signaling pathways of PR are involved in P4-regulated p53 expression in HUVECs.