Previous studies shows that connexins appear very early during murine embryo development, the gap junctional intercellular communication found in the inner cell mass of early embryo is also maintained in embryonic stem cells (ESC), and expression of oxytocin receptor (OTR) is developmentally regulated at early embryonic development. However, effect of oxytocin (OT) on the regulation of the connexin43 (Cx43) and maintenance of undifferentiation is not fully understood in stem cells. Therefore, we investigated the effect of OT on Cx43 expression and related signaling cascades in mouse ESC. OT increased Cx43 expression that was inhibited by the OTR inhibitor atosiban. In experiments to examine whether the effect of OT depends on lipid rafts, caveolin-1 (cav-1), cav-2, and flotillin-2, but not OTR, were detected in lipid raft fractions. Also, colocalization of OTR, cav-1, and cav-2 was not detected. Moreover, the lipid raft disruptor methyl-β-cyclodextrin did not attenuate OT-induced Cx43 expression. In experiments to examine related signaling pathways, OT activated cAMP/protein kinase A (PKA) which was inhibited by adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor PKI. OT increased nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) phosphorylation which was inhibited by PKI. OT also increased cAMP response element-binding (CREB)/CREB-binding protein (CBP) expression in the nucleus and induced the formation of CREB1/NF-κB/CBP complexes, which was blocked by the NF-κB-specific small interfering RNA, NF-κB inhibitors, SN50, and bay11-7082. Complex disruption by NF-κB inhibitors decreased OT-induced Cx43 expression. In conclusion, OT stimulates Cx43 expression through the NF-κB/CREB/CBP complex via the lipid raft-independent OTR-mediated cAMP/PKA in mouse ESC.