Diabetes mellitus is associated with a distinct cardiomyopathy. Whether cardiac myofilament function is altered in human diabetes mellitus is unknown. Myocardial biopsies were obtained from seven diabetic patients and five control, nondiabetic patients undergoing coronary artery bypass surgery. Myofilament function was assessed by determination of the developed force-Ca²⁺ concentration relation in skinned cardiac cells from flash-frozen human biopsies. Separate control experiments revealed that flash freezing of biopsy specimens did not affect myofilament function. All patients in the diabetes mellitus cohort were classified as Type 2 diabetes mellitus patients, and most showed signs of diastolic dysfunction. Diabetes mellitus was associated with depressed myofilament function, that is, decreased Ca²⁺ sensitivity (29%, P < 0.05 vs. control) and a trend toward reduction of maximum Ca²⁺-saturated force (29%, P = 0.08 vs. control). The slope of the force-Ca²⁺ concentration relation (Hill coefficient) was not affected by diabetes, however. We conclude that human diabetes mellitus is associated with decreased cardiac myofilament function. Depressed cardiac myofilament Ca²⁺ responsiveness may underlie the decreased ventricular function characteristic of human diabetic cardiomyopathy.