Runoff from acid sulfate soils (ASS) has severe environmental and economic impacts on estuarine ecosystems. Oysters display reduced abundance, growth rate and shell thickness when exposed to ASS runoff, yet the molecular underpinnings of their responses have not been explored. We hypothesised that the proteomes of wild Sydney rock oysters, Saccostrea glomerata, would differ between populations recurrently exposed to ASS compared with those unaffected by runoff from ASS. We used two-dimensional electrophoresis to compare protein abundances in the gills of S. glomerata collected from two sites close to (acidified) and two sites away from (reference) major ASS outflow drains in a south-east Australian estuary. Approximately 5% of the proteome was differentially expressed between oysters from acidified and reference sites, with five protein spots more abundant and one less abundant at the sites close to drains. Another protein spot was present only in oysters from reference sites. This study is the first screening of spatial variation in the protein expression of S. glomerata with respect to discharge from ASS. Altered protein expression may underpin short-term inducible responses to ASS runoff, or genetic resistance acquired through recurrent exposure of populations to the stressor.