Increased levels of O-linked attachment of N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins are implicated in the development of diabetic cardiomyopathy and are regulated by O-GlcNAc transferase (OGT) expression and its substrate UDP-GlcNAc. Therefore, the goal of this study was to determine whether the development of diabetes in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes, results in defects in cardiomyocyte mechanical function and, if so, whether this is associated with increased levels of O-GlcNAc and increased OGT expression. Six-week-old ZDF rats were hyperinsulinemic but normoglycemic, and there were no differences in cardiomyocyte mechanical function, UDP-GlcNAc, O-GlcNAc, or OGT compared with age-matched lean control rats. Cardiomyocytes isolated from 22-wk-old hyperglycemic ZDF rats exhibited significantly impaired relaxation, compared with both age-matched lean control and 6-wk-old ZDF groups. There was also a significant increase in O-GlcNAc levels in high-molecular-mass proteins in the 22-wk-old ZDF group compared with age-matched lean control and 6-wk-old ZDF groups; this was associated with increased UDP-GlcNAc levels but not increased OGT expression. Surprisingly, there was a significant decrease in overall O-GlcNAc levels between 6 and 22 wk of age in lean, ZDF, and Sprague-Dawley rats that was associated with decreased OGT expression. These results support the notion that an increase in O-GlcNAc on specific proteins may contribute to impaired cardiomyocyte function in diabetes. However, this study also indicates that in the heart the level of O-GlcNAc on proteins appears to be differentially regulated by age and diabetes.