Objective: Platelet activation by stimulatory factors leads to an increase in intracellular calcium concentration ([Ca2+](i)), which is essential for almost all platelet functions. Modulation of Ca(2+)influx and [Ca2+](i)in platelets has been emerging as a possible strategy for preventing and treating platelet-dependent thrombosis. Voltage-gated potassium 1.3 channels (Kv1.3) are highly expressed in platelets and able to regulate agonist-evoked [Ca2+](i)increase. However, the role of Kv1.3 channels in regulating platelet functions and thrombosis has not yet been elucidated. In addition, it is difficult to obtain a specific blocker for this channel, since Kv1.3 shares identical drug-binding sites with other K(+)channels. Here, we investigate whether specific blockade of Kv1.3 channels by monoclonal antibodies affects platelet functions and thrombosis. Approach and Results: In this study, we produced the anti-Kv1.3 monoclonal antibody 6E12#15, which could specifically recognize both human and mouse Kv1.3 proteins and sufficiently block Kv1.3 channel currents. We found Kv1.3 blockade by 6E12#15 inhibited platelet aggregation, adhesion, and activation upon agonist stimulation. In vivo treatment with 6E12#15 alleviated thrombus formation in a mesenteric arteriole thrombosis mouse model and protected mice from collagen/epinephrine-induced pulmonary thromboembolism. Furthermore, we observed Kv1.3 regulated platelet functions by modulating Ca(2+)influx and [Ca2+](i)elevation, and that this is mediated in part by P2X(1). Interestingly,Kv1.3(-/-)mice showed impaired platelet aggregation while displayed no abnormalities in in vivo thrombus formation. This phenomenon was related to more megakaryocytes and platelets produced inKv1.3(-/-)mice compared with wild-type mice. Conclusions: We showed specific inhibition of Kv1.3 by the novel monoclonal antibody 6E12#15 suppressed platelet functions and platelet-dependent thrombosis through modulating platelet [Ca2+](i)elevation. These results indicate that Kv1.3 could act as a promising therapeutic target for antiplatelet therapies.