This study examined the effects of different oxygenation levels and substrate availability on cardiac performance, metabolism, and biochemistry in sexually immature male and female rainbow trout (Oncorhynchus mykiss). Ventricle strips were electrically paced (0.5 Hz, 14°C) in hyperoxic or hypoxic Ringer solution. Our results demonstrate that 1) males sustain isometric force production (F) longer than females under hyperoxia (Po₂ = 640 mmHg) with exogenous glucose present; 2) contractility is not maintained under moderate (Po₂ = 130 mmHg) or severe hypoxia (Po₂ = 10–20 mmHg) with glucose in either sex; however, following reoxygenation, F is higher in females compared with males; and 3) female tissue has higher lactate levels, net lactate efflux, and lactate dehydrogenase activity than males, whereas males have higher glycogen, citrate synthase, and β-hydroxy acyl-CoA dehydrogenase activities, and greater inotropic responses to exogenous glucose and octanoate. No sex differences were detected in responsiveness to epinephrine and inhibitors of glucose transport or activities of hexokinase and pyruvate kinase. We conclude that sex differences exist in rainbow trout cardiac tissue: females appear to prefer glycolysis for ATP production, whereas males have a higher capacity for aerobic and lipid metabolism.