(1) Hyperglycemia leads to cytotoxicity in the heart. Although several theories are postulated for glucose toxicity-induced cardiomyocyte dysfunction, the precise mechanism still remains unclear. (2) This study was designed to evaluate the impact of elevated extracellular Ca(2+) on glucose toxicity-induced cardiac contractile and intracellular Ca(2+) anomalies as well as the mechanism(s) involved with a focus on Ca(2+)/calmodulin (CaM)-dependent kinase. Isolated adult rat cardiomyocytes were maintained in normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) media for 6-12 hours. Contractile indices were measured including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), and time-to-90% relengthening (TR(90)). (3) Cardiomyocytes maintained with HG displayed abnormal mechanical function including reduced PS, ±dL/dt, and prolonged TPS, TR(90) and intracellular Ca(2+) clearance. Expression of intracellular Ca(2+) regulatory proteins including SERCA2a, phospholamban and Na(+)-Ca(2+) exchanger were unaffected whereas SERCA activity was inhibited by HG. Interestingly, the HG-induced mechanical anomalies were abolished by elevated extracellular Ca(2+) (from 1.0 to 2.7 mM). Interestingly, the high extracellular Ca(2+)-induced beneficial effect against HG was abolished by the CaM kinase inhibitor KN93. (4) These data suggest that elevated extracellular Ca(2+) protects against glucose toxicity-induced cardiomyocyte contractile defects through a mechanism associated with CaM kinase.