OBJECTIVE: The large Ca and voltage-dependent potassium channel is important in regulating vascular tone in smooth muscle tissue. The rs11739136 KCNMB1 Glu65Lys polymorphism in the β1 subunit of the Ca and voltage-dependent potassium channel has, in some studies, been reported to associate with a protective effect on diastolic hypertension. The previous studies have, however, been conflicting, and the aim of the present study was to clarify the impact of the Glu65Lys polymorphism on hypertension at the population level of middle-aged people. DESIGN: Large-scale sex-stratified case-control studies and analyses of quantitative blood pressure. METHODS: The KCNMB1 Glu65Lys (rs11739136) polymorphism was genotyped in 5729 Danes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of PCR-generated primer extension products. RESULTS: In the population-based Inter99 cohort, the Glu65Lys polymorphism was associated with a 1.3% decrease in systolic blood pressure (P = 0.01) and a 1.1% decrease in diastolic blood pressure (P = 0.04) per Lys-allele among 2668 men. Among women, we observed no association between systolic or diastolic blood pressure and the Glu65Lys polymorphism. CONCLUSION: If replicated, our findings suggest that the KCNMB1 Glu65Lys polymorphism associates with reduced systolic and diastolic blood pressure in middle-aged men.