Here, we aimed to explore sex differences and the impact of sex hormones on cardiac contractile properties in doxorubicin (DOX)-induced cardiotoxicity. Male and female Sprague-Dawley rats were subjected to sham surgery or gonadectomy and then treated or untreated with DOX (2 mg/kg) every other week for 10 wk. Estrogen preserved maximum active tension (T_max) with DOX exposure, whereas progesterone and testosterone did not. The effects of sex hormones and DOX correlated with both altered myosin heavy chain isoform expression and myofilament protein oxidation, suggesting both as possible mechanisms. However, acute treatment with oxidative stress (H₂O₂) or a reducing agent (DTT) indicated that the effects on T_maxwere mediated by reversible myofilament oxidative modifications and not only changes in myosin heavy chain isoforms. There were also sex differences in the DOX impact on myofilament Ca²⁺sensitivity. DOX increased Ca²⁺sensitivity in male rats only in the absence of testosterone and in female rats only in the presence of estrogen. Conversely, DOX decreased Ca²⁺sensitivity in female rats in the absence of estrogen. In most instances, this mechanism was through altered phosphorylation of troponin I at Ser²³/Ser²⁴. However, there was an additional DOX-induced, estrogen-dependent, irreversible (by DTT) mechanism that altered Ca²⁺sensitivity. Our data demonstrate sex differences in cardiac contractile responses to chronic DOX treatment. We conclude that estrogen protects against chronic DOX treatment in the heart, preserving myofilament function.NEW & NOTEWORTHY We identified sex differences in cardiotoxic effects of chronic doxorubicin (DOX) exposure on myofilament function. Estrogen, but not testosterone, decreases DOX-induced oxidative modifications on myofilaments to preserve maximum active tension. In rats, DOX exposure increased Ca²⁺sensitivity in the presence of estrogen but decreased Ca²⁺sensitivity in the absence of estrogen. In male rats, the DOX-induced shift in Ca²⁺sensitivity involved troponin I phosphorylation; in female rats, this was through an estrogen-dependent mechanism.