O-GlcNAcylation was first found by Torres and Hart in monocytes. It is a dynamic and reversible post-translational modification catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation is increased in diabetic cardiomyopathy (DCM) patients and it has been reported that OGT plays an important role in the regulation of cardiac gene transcription, cell cycle and calcium homeostasis. The purpose of this study is to investigate the effects of OGT on signal transduction and function of β1-adrenoceptor (β1AR) in adult rat cardiomyocytes. We found that after overexpressing OGT by adenovirus vector in adult rat cardiomyocytes, cAMP formation and phosphorylation of phospholamban (PLB) at Ser16 (p16-PLB) were decreased under isoprenaline (ISO) stimulation. Over expression of OGT increased the intracellular [Ca]i and deteriorated the death of cardiomyocytes induced by prolonged stimulation with ISO. β1-adrenoceptor was overexpressed using a plasmid vector and then co-immunoprecipitation (co-IP) followed by Western blot was employed to define the O-GlcNAcylation of β1-adrenoceptor. The results showed that O-GlcNAcylation of β1-adrenoceptor was increased in OGT overexpressed cells, and there was no significant change in the formation of cAMP and phosphorylation of PLB after β1-adrenoceptor was blocked by CGP20712A. Given that OGT affects the signal transduction of β1-adrenoceptor in adult rat cardiomyocytes by increasing the O-GlcNAcylation of β1-adrenoceptor, the mechanism revealed in this study indicates that OGT and β1AR may be therapeutic targets in patients undergoing diabetic cardiomyopathy.