Diabetes mellitus impairs ventricular function, which itself may be disparately influenced by gender. This study compared the impact of gender on cardiac contractile response in ventricular myocytes from wild-type FVB and calmodulin-induced diabetic transgenic (OVE26) mice at young (2 month) and older (11 month) age. Mechanical and intracellular Ca2+ properties of cardiac myocytes were evaluated using an IonOptix MyoCam system. Diabetic mice of both genders exhibited significantly elevated blood glucose regardless of age. OVE26 myocytes displayed reduced peak shortening (PS) and maximal velocity of shortening/relengthening (+/- dL/dt), and prolonged time-to-PS (TPS) and time-to-90% relengthening (TR90), associated with higher resting intracellular Ca2+ levels and attenuated Ca(2+)-induced intracellular Ca2+ release compared with the FVB myocytes. Peak shortening and +/- dL/dt were smaller in female FVB groups when compared to the age-matched male counterparts. However, these gender differences were ablated by the diabetic state. No significant gender-related differences in intracellular Ca2+ handling were noted in either FVB or OVE26 myocytes, with the exception of overt gender differences in OVE26 mice when age was taken into account. Young female OVE26 mice exhibited better-preserved mechanical function while older female OVE26 mice displayed the worst mechanical function among all four OVE26 groups. In conclusion, our data confirmed impaired cardiac contractile function in diabetes, partially due to altered intracellular Ca2+ handling, in both genders. Mechanical differences existed between genders but were "cancelled off" by diabetic state. Nevertheless, a "female advantage" in ventricular function may still persist in young female diabetic subjects.