Aims: C-type Natriuretic Peptide (CNP) has a demonstrated hyperpolarizing effect on vascular smooth muscle cells. However, its autocrine function, including its electrophysiological effect on endothelial cells, is not known. Here, we report the effect of CNP on the membrane potential (E_m) of pulmonary microvascular endothelial cells and describe its target receptors, second messengers, and ion channels. Methods: We measured changes in E_m using fluorescence imaging and perforated patch-clamping techniques. In imaging experiments, samples were pre-incubated in the potentiometric dye, DiBAC4(3), and subsequently exposed to CNP in the presence of selective inhibitors of ion channels or second messengers. Results: CNP exposure induced a dose-dependent decrease in fluorescence, indicating that CNP induces endothelial cell hyperpolarization. CNP-induced hyperpolarization was inhibited by the K⁺ channel blockers, tetraethylammonium or iberiotoxin, the non-specific cation channel blocker, La³⁺, or by depletion or repletion of extracellular Ca²⁺ or K⁺, respectively. CNP-induced hyperpolarization was also blocked by pharmacological inhibition of protein kinase G (PKG) or by siRNA-mediated knockdown of the NPR-B receptor. CNP-induced hyperpolarization was mimicked by the PKG agonist, 8-Br-cGMP, and attenuated by both the nitric oxide synthase (eNOS) inhibitor, L-NAME, and the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Presence of iberiotoxin-sensitive, CNP-induced outward current was confirmed by perforated patch-clamping experiments. Conclusions: We conclude that CNP hyperpolarizes pulmonary microvascular endothelial cells by activating BK_Ca channels in a process mediated by the activation of NPR-B, PKG, eNOS, and sGC.