The voltage-gated proton channel Hv1 is expressed in various human cell types, including macrophages, epithelial cells, and sperm. Hv1 opening leads to proton efflux that alkalizes the cytosol. Here, we describe light-activated Hv1 inhibitors (photoswitches) that allow controlling its activity with high spatiotemporal precision. The photoswitches comprise a light-sensitive azobenzene moiety and 2-guanidinobenzimidazole (2GBI), a known Hv1 inhibitor. In the dark, photoGBI inhibits heterologously expressed Hv1 channels. Blue light, which isomerizes the azobenzene group from trans to cis conformation, releases inhibition. We demonstrate photocontrol of native proton currents in human macrophages and sperm using photoGBI, underlining their use as valuable optochemical tools to study the function of Hv1 channels.