High fructose intake has been linked to insulin resistance and cardiac pathology. Dietary fructose-induced myocardial signaling and morphological alterations have been described, but whether cardiomyocyte function is influenced by chronic high fructose intake is yet to be elucidated. The goal of this study was to evaluate the cardiomyocyte excitation-contraction coupling effects of high dietary fructose and determine the capacity for murine cardiomyocyte fructose transport. Male C57Bl/6J mice were fed a high fructose diet for 12 wk. Fructose- and control-fed mouse cardiomyocytes were isolated and loaded with the fura 2 Ca2+ fluorescent dye for analysis of twitch and Ca2+ transient characteristics (4 Hz stimulation, 37°C, 2 mM Ca2+). Myocardial Ca2+-handling protein expression was determined by Western blot. Gene expression of the fructose-specific transporter, GLUT5, in adult mouse cardiomyocytes was detected by real-time and conventional RT-PCR techniques. Diastolic Ca2+ and Ca2+ transient amplitude were decreased in isolated cardiomyocytes from fructose-fed mice relative to control (16 and 42%, respectively), coincident with an increase in the time constant of Ca2+ transient decay (24%). Dietary fructose increased the myofilament response to Ca2+ (as evidenced by a left shift in the shortening-Ca2+ phase loop). Protein expression of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), phosphorylated (P) phospholamban (Ser16), and P-phospholamban (Thr17) was reduced, and protein phosphatase 2A expression increased, in fructose-fed mouse hearts. Hypertension and cardiac hypertrophy were not evident. These findings demonstrate that fructose diet-associated myocardial insulin resistance induces profound disturbance of cardiomyocyte Ca2+ handling and responsiveness in the absence of altered systemic loading conditions.