Recent studies show significant effects of insulin and bioactive lipids on the intracellular Ca²⁺ handling of cardiomyocytes and these effects differ between normal and diabetic cardiomyocytes. In the present study we measured non-selective cation currents (NSCC) in isolated ventricular cardiomyocytes of adult wild-type (WT) and insulin resistant, obese ob/ob mice, a model of type 2 diabetes. Insulin activated an NSCC of similar magnitude in WT and ob/ob cardiomyocytes. Application of a membrane permeable DAG analogue (OAG) induced an NSCC, which was ~40% smaller in ob/ob than in WT cardiomyocytes. DAG can activate TRPC3 and TRPC6 and these proteins were expressed in the cardiomyocytes, though the expression was lower in ob/ob ventricles. Pretreatment with insulin increased the OAG-induced NSCC in WT (by ~50%) but not in ob/ob cells. Immunostaining demonstrated an insulin-induced translocation of TRPC3/6 to the plasma membrane in WT but not in ob/ob cardiomyocytes. In conclusion, insulin stimulated an NSCC that was not different between normal and diabetic cardiomyocytes, which suggests that it was not mediated via TRPC3/6. On the other hand, diabetic cardiomyocytes showed decreases in DAG-mediated NSCC, which may be due to decreased TRPC3 and TRPC6 expression and defective insulin-mediated trafficking of these proteins.