We demonstrated that normal cardiac myofilaments contain 32 total O-GlcNAcylation sites on MHC, Actin, MLC 1, MLC2 and Tn I and that exposure of skinned muscles to GlcNAc induces myofilament Ca2+ desensitization. Actin O-GlcNAcylation is increased in myofilaments following GlcNAc exposure and in two in vivo Diabetes Mellitus (DM) models. Yet, the mechanisms of O-GlcNAc-induced myofilament Ca2+ desensitization remain unclear. We investigated the effect of O-GlcNAc removal by an engineered hexosaminidase (CPJ) on Ca2+ sensitivity of skinned muscles from hearts of control and DM type 1 rats. We found that 1 hour exposure to CPJ reversed myofilament Ca2+ desensitization in DM cardiac muscles (EC50 4.17±0.48 µM pre-CPJ vs 2.73±0.22 µM post-CPJ, n=5 vs n=4, p=0.029 ), but had no effect in control muscles (EC50 2.73±0.17 µM pre-CPJ vs 2.6±0.15 µM post-CPJ, n=6 vs n=5, p=n.s). CPJ activity against O-GlcNAc was verified by western blot on treated lysates. These results suggest that in diabetic muscle O-GlcNAcylation directly affects myofilament Ca2+ sensitivity. To address potential mechanism(s), we characterized O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) protein interactions in fresh whole heart homogenates from both groups by immunoprecipitation followed by Western blotting. Remarkably, we observed OGT and OGA interactions with members of thick (MyBP-C, MLC1, MLC2) and thin (Actin, TnI, Tm) myofilament proteins, which were differentially increased with actin, Tm and MLC1 in DM rat homogenates compared to control (n=4 vs n=4, p< 0.05). These results strongly suggest that in DM a specific increase in O-GlcNAcylation of cardiac myofilament proteins, and in OGT/OGA association with actin, tropomyosin and MLC1 leads to dysfunctional regulation of myocardial contractility.