Mercury enters marine food webs in the form of microbially generated monomethylmercury. Microbial methylation of inorganic mercury, generating monomethylmercury, is widespread in low-oxygen coastal sediments. The degree to which microbes also methylate mercury in the open ocean has remained uncertain, however. Here, we present measurements of the stable isotopic composition of mercury in nine species of marine fish that feed at different depths in the central North Pacific Subtropical Gyre. We document a systematic decline in δ202Hg, Δ199Hg and Δ201Hg values with the depth at which fish feed. We show that these mercury isotope trends can be explained only if monomethylmercury is produced below the surface mixed layer, including in the underlying oxygen minimum zone, that is, between 50 and more than 400 m depth. Specifically, we estimate that about 20–40% of the monomethylmercury detected below the surface mixed layer originates from the surface and enters deeper waters either attached to sinking particles, or in zooplankton and micronekton that migrate to depth. We suggest that the remaining monomethylmercury found at depth is produced below the surface mixed layer by methylating microbes that live on sinking particles. We suggest that microbial production of monomethylmercury below the surface mixed later contributes significantly to anthropogenic mercury uptake into marine food webs.