Adriamycin (ADR) is a potent anticancer drug, but its use is limited by a dose-dependent cardiotoxicity. Oxidative stress is regarded as the mediating mechanism of ADR cardiotoxicity. However, cardiac proteins that are oxidatively modified have not been well characterized. We took a redox proteomics approach to identify increasingly oxidized murine cardiac proteins after a single injection of ADR (ip, 20 mg/kg body wt). The specific carbonyl levels of three proteins were significantly increased, and these proteins were identified as triose phosphate isomerase (TPI), beta-enolase, and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). TPI and enolase are key enzymes in the glycolytic pathway, and ETF-QO serves as the transporter for electrons derived from a variety of oxidative processes to the mitochondria respiratory chain. Cardiac enolase activity in ADR-treated mice was reduced by 25%, whereas the cardiac TPI activity remained unchanged. Oxidation of purified enolase or TPI via Fenton chemistry led to a 17 or 23% loss of activity, respectively, confirming that a loss of activity was the consequence of oxidation. The observation that these cardiac enzymes involved in energy production are more oxidized resulting from ADR treatment indicates that the bioenergetic pathway is an important target in ADR-initiated oxidative stress.