Activity of voltage-gated K⁺ (Kv) channels controls membrane potential (E_m). Membrane depolarization due to blockade of K⁺ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E_m in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel - and -subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kv1.1, Kv2.1, and Kv3), but only MASMC expressed voltage-dependent Ca²⁺ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E_m depolarization in both MASMC and IEC. The 4-AP-induced E_m depolarization increased [Ca²⁺]_cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel - and -subunits. The function of these Kv channels plays an important role in controlling E_m. The membrane depolarization-mediated increase in [Ca²⁺]_cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.