We test the hypothesis that Colorado River flow is important in providing nursery habitat for the Gulf corvina (Cynoscion othonopterus), a commercially valuable and endemic fish in the upper Gulf of California. We use oxygen isotopes in otoliths to determine when these fish inhabit isotopically different bodies of water (Gulf of California versus the Colorado Estuary). The δ18O values in the natal otoliths of C. othonopterus, significantly more negative than can be predicted by temperature alone, provide evidence that this species uses the brackish habitats created by flow of the Colorado River. A significant log-linear relationship between the natal δ18O values and the cumulative flow of the Colorado River during natal development confirms use of brackish habitat in years that the Colorado River water reached the Gulf. Natal δ18O values indicate that C. othonopterus seek out estuarine habitats with salinities between 26 and 38. Reduction in Colorado River flow since the construction of upstream dams has reduced the size of nursery habitat for C. othonopterus. Our results support the hypothesis that declines in commercial landings can be at least partially attributed to reduced river flow. Increased flow would increase nursery habitat and likely benefit recruitment.