Proton transfer across single-layer graphene proceeds with large computed energy barriers and is therefore thought to be unfavourable at room temperature unless nanoscale holes or dopants are introduced, or a potential bias is applied. Here we subject single-layer graphene supported on fused silica to cycles of high and low pH, and show that protons transfer reversibly from the aqueous phase through the graphene to the other side where they undergo acid–base chemistry with the silica hydroxyl groups. After ruling out diffusion through macroscopic pinholes, the protons are found to transfer through rare, naturally occurring atomic defects. Computer simulations reveal low energy barriers of 0.61–0.75 eV for aqueous proton transfer across hydroxyl-terminated atomic defects that participate in a Grotthuss-type relay, while pyrylium-like ether terminations shut down proton exchange. Unfavourable energy barriers to helium and hydrogen transfer indicate the process is selective for aqueous protons.