Several members of the EPH kinase family and their ligands are involved in blood pressure regulation, and such regulation is often sex- or sex hormone-dependent, based on animal and human genetic studies. EPHB6 gene knockout (KO) in mice leads to hypertension in castrated males but not in un-manipulated KO males or females. To assess whether this finding in mice is relevant to human hypertension, we conducted a human genetic study for the association of EPHB6 and its two ligands, EFNB1 and EFNB3, with hypertension in hypogonadic patients. Seven hundred and fifty hypertensive and 750 normotensive Han Chinese patients, all of whom were hypogonadic, were genotyped for single nucleotide polymorphisms (SNPs) within the regions of the genes, plus an additional 50 kb 5' of the genes for EPHB6, EFNB1 and EFNB3. An imputed insertion/deletion polymorphism, rs35530071, was found to be associated with hypertension at p-values below the Bonferroni-corrected significance level of 0.0024. This marker is located 5' upstream of the EFNB3 gene start site. Previous animal studies showed that while male EFNB3 gene knockout mice were normotensive, castration of these mice resulted in hypertension, corroborating the results of the human genetic study. Considering the significant associations of EFNB3 SNPs with hypertension in hypogonadic males and supporting evidence from castrated EFNB3 KO mice, we conclude that loss-of-function variants of molecules in the EPHB6 signaling pathway in the presence of testosterone are protective against hypertension in humans.