Essential hypertension involves a gradual and sustained increase in total peripheral resistance reflecting an increased vascular tone, which is associated with a depolarization of vascular smooth muscle cells (VSMCs) and relies on a change in the expression profile of voltage-dependent ion channels (mainly Cav and Kv channels) that promotes arterial contraction. Kv channels are principal contributors to determine the resting membrane potential of VSMCs. However, the changes in their expression and/or modulation during hypertension are poorly defined, mainly due to their large molecular diversity. Here we have undertaken the study of the molecular and functional characterization of Kv and KCa channels in VSMCs in essential hypertension by using VSMCs from mesenteric arteries obtained from a hypertensive inbred mouse strain, BPH, and the corresponding normotensive strain, BPN. Mice were killed by decapitation after isofluorane anesthesia. Real-time PCR using low-density taqman® arrays revealed a differential distribution of K⁺ channel subunits mRNA between both strains, the most conspicuous change being the “de novo” expression of Kcng3 in hypertensive phenotype. To study the functional relevance of this change, we characterized the electrophysiological properties of freshly dissociated VSMCs of BPN and BPH mice with the patch-clamp technique. Both KCa current density, (500 nM paxilline-sensitive current) and Kv current density (paxilline-resistant current) were significantly smaller in BPH cells at all voltages. Application of selective Kv2 blockers as 50 nM stromatoxin or 20 nM guangxitoxin induced a reduction of the current amplitude that was significantly smaller in BPH cells, suggesting that the reduction in the functional expression of Kv2 currents in BPH cells contribute to the decreased Kv current in these cells. These findings could be explained by the “de novo” expression of Kv6.3 mRNA in BPH VSMCs, as Kv6.3 subunits associate with Kv2 channels to form heteromultimers with decreased current amplitude. In agreement with this, Kv6.3 protein could be detected by western-blot in BPH but not in BPN arteries. Moreover, intracellular application of anti-Kv6.3 antibody decreased Kv current amplitude in BPH VSMCs but had no effect in BPN ones, demonstrating a functional role of this subunit in hypertensive VSMCs. These data suggest that the expression of Kv6.3 channels in resistance vessels could contribute to the natural development of hypertension, and can help to understand the molecular basis of this complex and multifactorial disease.