Objective To establish an endothelial conditional connexin 43 (Cx43)-knockout mouse model and use it to examine vascular relaxation / constriction. Methods Eight Cx43flox / flox mice were crossed with C57BL/ 6 wild type (WT) mice and the offspring were backcrossed with C57BL/ 6 mice. This backcross process was repeated nine times, after which the Cx43flox / + mice were crossed with vascular endothelial-cell-specific Tie2-Cre recombinase mice to obtain Tie2-Cre /Cx43flox / + mice. The genotype was confirmed by mouse tail direct polymerase chain reaction (PCR). Expression of Cx43 in the superior mesenteric artery ( SMA) was measured by Western Blot and immunofluorescence. SMA rings were used to measure vascular relaxation and the contractile response to acetylcholine (ACh) and norepinephrine. Results Tie2-Cre /Cx43flox / +offspring were obtained and identified by PCR, Western Blot and immunofluorescence. Cx43 expression and AChinduced endothelium-dependent relaxation reactivity of the SMA were significantly decreased in Tie2-Cre / Cx43flox / + mice compared with WT mice ( P < 0. 01). Conclusions A mouse model with endothelium-specific Cx43-knockout was successfully established by intercrossing Tie2-Cre mice with Cx43^{flox / flox} mice. Cx43 expression in vascular tissues and the relaxation reactivity of SMA were decreased by conditional Cx43 knockout. These mice may thus provide an animal model for studying the link between Cx43 and vascular function.