Background Patients treated for breast cancer have a high incidence of cardiovascular complications. In this study, we evaluated the impact of breast cancer on cardiac function and cardiomyocyte Ca-handling protein expression. We also investigated whether exercise training (ET) would prevent these potential alterations. Methods and Results Transgenic mice with spontaneous breast cancer (mouse mammary tumor virus-polyomavirus middle T antigen [MMTV-PyMT+], n=15) and littermate mice with no cancer (MMTV-PyMT-, n=14) were studied. For the ET analysis, MMTV-PyMT+ were divided into sedentary (n=10) and exercise-trained (n=12) groups. Cardiac function was evaluated by echocardiography with speckle-tracking imaging. Exercise tolerance test was conducted on a treadmill. Both studies were performed when the tumor became palpable and when it reached 1 cm. After euthanasia, Ca-handling protein expression (Western blot) was evaluated. Exercise capacity was reduced in MMTV-PyMT+ compared with MMTV-PyMT- (=0.031). Longitudinal strain ( <0.001) and strain rate (=0.030) were impaired. Cardiomyocyte phospholamban was increased (=0.011), whereas phospho-phospholamban and sodium/calcium exchanger were decreased (=0.038 and =0.017, respectively) in MMTV-PyMT+. No significant difference in sarcoplasmic or endoplasmic reticulum calcium 2 ATPase (SERCA2a) was found. SERCA2a/phospholamban ratio was reduced (=0.007). ET was not associated with increased exercise capacity. ET decreased left ventricular end-systolic diameter (=0.038) and end-diastolic volume (=0.026). Other morphological and functional cardiac parameters were not improved by ET in MMTV-PyMT+. ET did not improve cardiomyocyte Ca-handling protein expression. Conclusions Breast cancer is associated with decreased exercise capacity and subclinical left ventricular dysfunction in MMTV-PyMT+, which is at least partly associated with dysregulation of cardiomyocyte Ca handling. ET did not prevent or reverse these changes.