We have reported previously that hearts from type 2 diabetic (db/db) mice show decreased cardiac efficiency due to increased work-independent myocardial oxygen consumption (unloaded MVO₂), indicating higher oxygen use for non-mechanical processes such as basal metabolism (MVO_2BM) and excitation-contraction coupling (MVO_2ECC). Although alterations in cardiac metabolism and/or Ca²⁺ handling may contribute to increased energy expenditure in diabetic hearts, direct measurements of the oxygen cost for these individual processes have not been determined. In this study we (i) validate a procedure for measuring unloaded MVO₂ directly (MVO_2unloaded) and for determining MVO_2BM and MVO_2ECC separately in isolated perfused mouse hearts, and (ii) determine oxygen cost for these processes in hearts from db/db mice. Unloaded MVO₂, extrapolated from the relationship between cardiac work (measured as pressure-volume area, PVA) and MVO₂ was found to correspond with MVO₂ measured directly in unloaded retrograde perfused hearts (MVO_2unloaded). MVO₂ in K⁺-arrested hearts was defined as MVO_2BM; the difference between MVO_2unloaded and MVO_2BM represented MVO_2ECC. This procedure was validated by demonstrating that elevations in perfusate FA and/or Ca²⁺ concentrations resulted in changes in either MVO_2BM and/or MVO_2ECC. The higher MVO_2unloaded in db/db mice was due to both a higher MVO_2BM and MVO_2ECC. Elevation of glucose and insulin decreased FA oxidation and reduced both MVO_2unloaded and MVO_2BM. In conclusion, this study provides direct evidence that MVO_2BM and MVO_2ECC are elevated in diabetes, and that acute metabolic interventions can have a therapeutic benefit in diabetic hearts due to a MVO₂-lowering effect.