In this study, field biotelemetry and laboratory physiology approaches were coupled to allow understanding of the behavioral and physiological responses of fish to winter hypoxia. The biotelemetry study compared dissolved oxygen levels measured throughout the winter period with continually tracked locations of nine adult largemouth bass obtained from a whole‐lake submerged telemetry array. Fish habitat usage was compared with habitat availability to assess whether fish were selecting for specific dissolved oxygen concentrations. The laboratory study examined behavioral and physiological responses to progressive hypoxia in juvenile largemouth bass acclimated to winter temperatures. Results from the dissolved oxygen measurements made during the biotelemetry study showed high variance in under‐ice dissolved oxygen levels. Avoidance of water with dissolved oxygen <2.0 mg/L by telemetered fish was demonstrated, but significant use of water with intermediate dissolved oxygen levels was also found. Results from the lab experiments showed marked changes in behavior (i.e., yawning and vertical movement) at <2.0 mg/L of dissolved oxygen but no change in tissue lactate, an indicator of anaerobic metabolism. Combined results of the biotelemetry and laboratory studies demonstrate that a dissolved oxygen content of 2.0 mg/L may be a critical threshold that induces behavioral responses by largemouth bass during the winter. In addition, the use by fish of areas with intermediate levels of dissolved oxygen suggests that there are multiple environmental factors influencing winter behavior.