The primary features of type 2 diabetes (T2D) are both insulin resistance and impaired beta cell function. Abnormal glucose handling and variable degrees of peripheral insulin resistance characterise T2D. Skeletal muscle is the largest insulin-regulated glucose sink in the body and represents approximately 80% of whole body insulin-stimulated glucose uptake. As a result a fundamental aspect of T2D is abnormal glucose disposal in contractile tissues triggering metabolic dysregulation and glucotoxic side effects. The significance of skeletal muscle to T2D has prompted research into the perturbed glucose handling mechanisms in suitable animal models, such as muscle tissues from the spontaneously diabetic Goto-Kakizaki rat. Highly sensitive protein analysis techniques such as fluorescence difference in-gel electrophoresis (DIGE) and electrospray ionization liquid chromatography mass spectrometry (ESI LC/MS) were employed to analysis the diabetic muscle tissue proteome and sub-proteome. A differential expression pattern was observed for proteins involved in glycolysis, the citric acid cycle, oxidative phosphorylation, lipolytic catabolism, the contractile apparatus, cellular detoxification mechanisms and the stress response. These findings demonstrate a perturbed protein expression pattern in diabetic skeletal muscle, which reflects underlying molecular alterations. Over all this study has supplied several potential biomarkers of T2D and adds further knowledge to the mechanism of a complex metabolic disorder.