Diabetes is characterized by high blood glucose level termed hyperglycemia affecting skeletal muscle structure and function by an unclear molecular mechanism. This study aimed to investigate the effect and underlying mechanism(s) of hyperglycemia on skeletal muscle both in vitro and in vivo. Treatment with hyperglycemic condition (25 mM) for 48 h significantly inhibited C2C12 myoblast proliferation detected by MTT assay whilst flow cytometry revealed an interruption of the cell cycle at subG1 and G2/M phases. An exposure to hyperglycemic condition significantly decreased the myosin heavy chain (MHC) protein expression in the differentiated myotube and tibialis anterior (TA) muscle of Wistar rats. In addition, the muscle cross-section area (MCA) of TA muscle in diabetic rats were significantly decreased compared to the non-diabetic control. Western blotting analysis of C2C12 myoblasts and differentiated myotubes revealed the increased expressions of cleaved-caspase-9 and cleaved-caspase-3, but not cleaved-caspase-8. Of note, these caspases in the TA muscles were not changed under hyperglycemic condition. Quantitative real-time polymerase chain reaction (qRT-PCR) of the hyperglycemic myoblasts and TA muscles revealed modulation of the gene expression of sirtuins (SIRTs). In C2C12 myoblasts, the expressions of SIRT1, SIRT2 and SIRT4 were upregulated whilst SIRT7 was downregulated. Meanwhile, the expressions of SIRT1, SIRT2 in TA muscles were upregulated whilst SIRT4 was downregulated. Taken together, this study showed that hyperglycemia induced cell cycle arrest and apoptosis in myoblasts, and protein degradation and atrophy in skeletal muscle most likely via modulation of SIRTs gene expression.