Aerobic exercise training is known to have profound cardioprotective effects in disease, yet cellular mechanisms remain largely undefined. We tested the hypothesis that increased sarcoplasmic reticulum Ca²⁺ buffering and increased voltage-gated Ca²⁺ channel density underlie coronary smooth muscle intracellular Ca²⁺ (Ca²⁺_i) dysregulation in diabetic dyslipidemia and that exercise training would prevent these increases. Yucatan swine were maintained: 1) control, 2) alloxan-induced hyperglycemic, 3) high fat/cholesterol fed, 4) hyperglycemic plus high fat/cholesterol fed (diabetic dyslipidemic), and 5) diabetic dyslipidemic plus exercise-trained (treadmill running). After 20 wks, the heart was removed and smooth muscle cells isolated from the right coronary artery. We utilized fura-2 imaging of Ca²⁺_i levels to separate the functional role of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) from the Na⁺-Ca²⁺ exchanger (NCX) and the plasmalemmal Ca²⁺-ATPase (PMCA), and whole-cell patch clamp to examine voltage-gated Ca²⁺ channel current density (i.e. Ca²⁺ influx). Results indicated that diabetic dyslipidemia impaired plasmalemmal Ca²⁺ efflux, increased basal Ca²⁺_i levels, increased SERCA protein and sarcoplasmic reticulum Ca²⁺_i buffering, and elicited an ~50% decrease in voltage-gated Ca²⁺-channel current density. Exercise training concurrent with the diabetic dyslipidemic state restored plasmalemmal Ca²⁺ efflux, SERCA protein, sarcoplasmic reticulum Ca²⁺_i buffering, and voltage-gated Ca²⁺-channel current density to control levels. Interestingly, basal Ca²⁺_i levels were significantly lower in the exercise-trained group compared to control. Collectively, these results demonstrate a crucial role for exercise in the prevention of diabetic dyslipidemia-induced Ca²⁺_i dysregulation.