Otopetrins (Otop1-Otop3) belong to a newly identified family of proton (H) channels activated by extracellular acidification. Here, we found that Zn activates the mouse Otop3 (mOtop3) proton channels by using electrophysiological patch-clamp techniques. In mOtop3-expressing human embryonic kidney HEK293T cells, a biphasic inward mOtop3 H current comprising a fast transient current followed by a sustained current was observed upon extracellular acidification at pH 5.0. No significant activation of the mOtop3 channel was observed at pH 6.5 and 7.4, but interestingly, Zn dose-dependently induced a sustained activation of mOtop3 under these pH conditions. Increasing the Zn concentration had no effect on the reversal potential of the channel currents, suggesting that Zn does not permeate through the mOtop3. The activation of the mOtop3 channel was specific to Zn among divalent metal cations. Our findings reveal a novel modulatory mechanism of mOtop3 proton channels by Zn.