Human vascular connexins (Cx37, Cx40, Cx43, and Cx45) can form various types of gap junction channels to synchronize vasodilation/constriction to control local circulation. Most of our knowledge on heterotypic gap junctions of these vascular connexins was from studies on rodent connexins. In human vasculature, the same four homolog connexins exist, but whether these human connexins can form heterotypic GJs as those of rodents have not been fully studied. Here we used in vitro expression system to study the coupling status and GJ channel properties of human heterotypic Cx37/Cx40, Cx37/Cx43, and Cx37/Cx45 GJs. Our results showed that Cx37/Cx43 and Cx37/Cx45 GJs, but not Cx37/Cx40 GJs, were functional and each with unique rectifying channel properties. The failure of docking between Cx37 and Cx40 could be rescued by designed Cx40 variants. Characterization of the heterotypic Cx37/Cx43 and Cx37/Cx45 GJs may help us in understanding the intercellular communication at the myoendothelial junction.