OLETF rat is a model of spontaneous non-insulin-dependent diabetes mellitus (DM), accompanying diastolic dysfunction associated with abnormal Ca²⁺ handling and decrease in sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) expression. The aim of this study was to examine whether SERCA2a gene transfer can restore left ventricular (LV) function in this DM in terms of LV mechanical work and energetics. DM rats were randomized to receive adenovirus carrying SERCA 2a gene (DM+Serca) or β-galactosidase (DM+Gal), or saline (DM+Sa) by catheter-based (cross-clamping) technique. LV mechanoenergetics was assessed in cross-circulated whole heart preparations 3 days after the infection. In DM, end-systolic pressure (ESP_0.1) was low and end-diastolic pressure (EDP_0.1) was high at 0.1 ml intraballoon water. In DM+Serca, however, ESP_0.1 increased and EDP_0.1 decreased. LV relaxation rate in DM+Serca was faster than that of DM+Gal/Sa groups. Oxygen cost of LV contractility in DM was higher than that of normal, indicating energy wasting in Ca²⁺ handling during E-C coupling. The O₂ cost of LV contractility decreased to non-DM rat level in DM+Serca, although it remained high in DM+Gal and DM+Sa. These results indicate that SERCA2a overexpression by SERCA2a gene transfer improves not only LV mechanics but also energetics in DM rat hearts. [J Physiol Sci. 2006;56 Suppl:S54]