Data from animal experiments strongly suggest that ceramide is an important mediator of lipotoxicity in the heart and that accumulation of ceramide contributes to cardiomyocyte apoptosis associated with type 2 diabetes and obesity. However, it remains unknown whether a similar relationship is present also in the human heart. Therefore, we aimed to examine whether myocardial apoptosis in obese and type 2 diabetic patients is associated with elevated ceramide level. The study included 11 lean and 26 overweight or moderately obese subjects without (n = 11, OWT) or with (n = 15, T2D-OWT) a history of type 2 diabetes. Samples of the right atrial appendage were obtained from patients at the time of coronary bypass surgery. Compared with lean subjects, the extent of DNA fragmentation (a marker of apoptosis) was significantly higher in the myocardium of OWT patients and increased further in T2D-OWT subjects. However, the content of ceramide and sphingoid bases remained stable. Interestingly, the mRNA level of enzymes involved in synthesis and degradation of ceramide including serine palmitoyltransferase, sphingosine kinase 1, neutral sphingomyelinase, and ceramidases was markedly higher in the myocardium of OWT and T2D-OWT patients compared with lean subjects. Our results indicate that in the human heart, or at least in the atrium, ceramide is not a major factor in cardiomyocyte apoptosis associated with obesity and type 2 diabetes.