Oxygen isotope values from lacustrine carbonate in the Laney Member of the Green River Formation (Wyoming) exhibit a sudden, basinwide, ∼6‰ upsection decrease in δ18O at ca. 49 Ma. 40Ar/39Ar geochronology constrains the duration of the isotopic shift to ≤∼200,000 years. This change coincides with a sudden change in lake type, from balanced-filled in the lower LaClede Bed to overfilled in the upper LaClede Bed, as well as an increase in the proportion of calcitic (>80% calcite out of total carbonate by X-ray diffraction [XRD]) samples from 32% to 73%. The δ18O shift is correlatable through several locations across the Greater Green River Basin, and also coincides with a previously observed shift to less radiogenic 87Sr/86Sr. Minimum δ18O values observed are the same as values previously reported in aragonitic bivalves from the same unit, indicating that low δ18O in this record is not diagenetic. A simultaneous shift to evaporative conditions in the Uinta Basin to the south indicates that the δ18O shift and lake-type change are not driven by regional climatic cooling and/or humidity increase. We propose that all of these observations resulted from the capture by Lake Gosiute of a river that drained higher elevations in central or north-central Idaho. Mass-balance modeling of Eocene Lake Gosiute indicates that capture of a river with an annual average discharge of ∼20 billion m3/a (slightly larger than the modern Snake River) and δ18O of –24‰ standard mean ocean water (SMOW) or lower would be capable of producing the observed change. A more likely alternative is a river with less negative δ18O and greater discharge. For example, if river waters had a δ18O composition of ∼−16‰, an estimated river discharge of ≥50 billion m3/a would produce the same effect.