This investigation examined microRNA-208a (miR-208a) as a potential biomarker of isoproterenol (ISO)-induced cardiac injury in superoxide dismutase-2 (Sod2^+/−) and the wild-type mice, and the potential sensitivity of Sod2^+/− mice to ISO-induced toxicity. A single intraperitoneal injection of ISO was administered to age-matched wild-type and Sod2^+/− mice at 0, 80, or 160 mg/kg. Plasma miR-208a, cardiac troponin I (cTnI), and ISO systemic exposure were measured at various time points postdose. Hearts were collected for histopathology examination and for tissue expression of miR-208a and myosin heavy chain 7. ISO administration caused increases in cTnI and miR-208a plasma levels that correlated with myocardial damage; however, the magnitude of increase differed according to the types of mice. At similar ISO systemic exposure, the magnitude of cTnI was greater in wild-type mice compared to Sod2^+/− mice; however, the magnitude of miR-208a was greater in Sod2^+/− mice than that of the wild-type mice. Myocardial degeneration occurred at ≥3 hr in the wild-type and ≥6 hr in Sod2^+/− mice. At ≥24 hr after ISO administration, miR-208a appeared superior to cTnI in indicating myocardial injury in both wild-type and Sod2^+/− mice. Sod2^+/− mice were not more sensitive than wild-type mice to ISO-induced toxicity.