Left ventricle diastolic dysfunction (LVDD), characterized by slow or incomplete relaxation of the ventricle during diastole, is an important contributor to heart failure development in diabetic patients. LVDD can be found in pre-diabetic patients as well as in almost half of asymptomatic patients with well-controlled diabetes. Reduced Ca 2+ reuptake into the sarcoplasmic reticulum (SR) by the SR Ca 2+ ATPase (SERCA) is an important contributor to the slow relaxation observed in diastolic dysfunction. Despite this there are still conflicting results with respect to SERCA expression and function in animal models of diabetes, due perhaps to differences in the duration and severity of diabetes and the presence of systemic complications that can per se alter SERCA expression and/or function. Here, we tested the hypothesis that the impaired relaxation observed in diabetic mice is a consequence of the direct effect of insulin on SERCA. We measured Ca 2+ transient relaxation in isolated cardiomyocytes from 8 weeks cardiomyocyte-selective insulin receptor knockout (CIRKO) mice (Cre-IR lox / lox ), , which lack systemic complications, and compared them with their WT littermates (IR lox / lox ) . Cardiomyocytes loaded with the Ca 2+ dye Fluo-4 were field stimulated at 2 and 3.3 Hz, at 37°C. The intracellular Ca 2+ was monitored using a custom-made epifluorescence system. SERCA and Na + /Ca 2+ exchanger (NCX) expression level was reported as the fold change relative to WT. We found that Ca 2+ transient relaxation was prolonged at both frequencies in CIRKO compared with WT, while retaining a normal frequency-dependent acceleration of relaxation. The exponential time constants were 104±4 (WT) vs. 190±16 (CK) ms at 3.3 Hz, n= 20/group, p<0.001 and 111±6 (WT) vs. 226±19 (CK) ms at 2 Hz, n= 20/group, p<0.001). We also found a reduction in SERCA expression (mRNA: 0.65±0.03, n=10, p<0.001; protein:0.62±0.11, n=4, p<0.05) with no difference in NCX expression (mRNA: 0.92±0.06 n=10/group, p=0.5; protein 0.96±0.05, n=6/group, p=0.2). In conclusion, our findings suggest that the slower Ca 2+ removal observed in the systemic models of diabetes is a direct effect of insulin on SERCA, which could contribute to the LVDD observed in diabetic patients.