Due to the substantial photosynthetic biomass in their habitat, salmonids such as the rainbow trout (Oncorhynchus mykiss) can be subject to hyperoxia in addition to high temperatures associated with climate change. Both stressful conditions increase the incidence of damaging reactive oxygen species (ROS). The mitochondrial association of hexokinase has been shown to increase in the hearts of certain fish experiencing hypoxia in a putative cardioprotective response to oxidative stress. In this study, the mitochondrial association of hexokinase I (HKI) and markers of oxidative damage and metabolic stress were probed to elucidate the cardioprotective role of hexokinase in the rainbow trout. Results showed that the co-administration of hyperoxia and hyperthermia increased the ventricular mitochondrially-bound fraction of HKI, whereas exposure to hyperthermia in normoxia had no effect; in the combined condition there was little evidence of increased stress. A second in vitro study using ventricular strips and isolated cardiomyocytes was undertaken to reconcile the cardioprotective role of HK in the rainbow trout with findings in mammalian studies, confirming that mitochondrial association of HK maintains aerobic efficiency and inhibits apoptosis. Finally, protein sequence analysis suggested that the physiological contributions of HKI and HKII in salmonids vary from those in mammals, further explaining the dynamic nature of the traditionally-inert HKI. Together, these findings help to explain the broader functions of HKI in the salmonid heart, and illustrate the role of complex environmental conditions in defining physiological responses.