Selenium (Se) undergoes redox transformations and isotopic fractionations at relatively high redox potentials and could therefore provide insight into changes in oceanic and atmospheric O2 levels over Earth’s history. We test this idea with Se data from the 2.5 Ga Mount McRae Shale (Hamersley Basin, Australia), which records temporary enrichments in abundances and isotopes of other redox-sensitive elements indicating a “whiff of oxygen” in Earth’s atmosphere before the Great Oxidation Event. Se isotopic ratios expressed as δ82/78Se and abundances relative to crustal background show significant positive excursions of up to 1.1‰ and an enrichment 13 times above background, respectively, overlapping with excursions in Mo and N isotopes and abundances. Because Se has a relatively high redox potential and photosynthetic oxidation pathways are unknown, our data thus suggest that Se was mobilized by free O2 during this interval. The isotopic fractionation likely occurred during transport of Se oxyanions from the site of weathering to the outer shelf. Although O2 could have been produced locally on land and may not necessarily have increased in the global atmosphere, our results strengthen the inference of an early origin of oxygenic photosynthesis long before the Paleoproterozoic Great Oxidation Event. This is the first report of a Se isotope excursion in the Precambrian rock record, and it confirms that Se isotopes can serve as a useful redox proxy in deep time.