Multiple lines of evidence indicate that serotonin (5-hydroxytryptamine [5-HT]) and voltage-gated K⁺ (K_V) channels play a central role in the pathogenesis of pulmonary hypertension (PH). We hypothesized that 5-HT might modulate the activity of K_V channels, therefore establishing a link between these pathogenetic factors in PH. Here, we studied the effects of 5-HT on K_V channels present in rat pulmonary artery smooth muscle cells (PASMC) and on hK_V1.5 channels stably expressed in Ltk⁻ cells. 5-HT reduced native K_V and hK_V1.5 currents, depolarized cell membrane, and caused a contraction of isolated pulmonary arteries. The effects of 5-HT on K_V currents and contraction were markedly prevented by the 5-HT_2A receptor antagonist ketanserin. Incubation with inhibitors of phospholipase C (U73122), classic protein kinase Cs (Gö6976), or tyrosine kinases (genistein and tyrphostin 23), the cholesterol depletion agent β-cyclodextrin or concanavalin A, an inhibitor of endocytotic processes, also prevented the effects of 5-HT. In homogenates from pulmonary arteries, 5-HT_2A receptors and caveolin-1 coimmunoprecipitated with K_V1.5 channels, and this was increased on stimulation with 5-HT. Moreover, K_V1.5 channels were internalized when cells were stimulated with 5-HT, and this was prevented by concanavalin A. These findings indicate that activation of 5-HT_2A receptors inhibits native K_V and hK_V1.5 currents via phospholipase C, protein kinase C, tyrosine kinase, and a caveolae pathway. K_V channel inhibition accounts, at least partly, for 5-HT-induced pulmonary vasoconstriction and might play a role in PH.