Lysine acetylation is an important post-translational modification, directly affecting signalling pathways that regulate metabolic function. The acetyltransferase, general control of amino acid synthesis 5 (GCN5) has been proposed as a key enzyme regulating muscle insulin action and metabolism via its ability to acetylate and inhibit peroxisome proliferator activated receptor coactivator 1α (PGC-1α). However, the specific role of GCN5 in regulating skeletal muscle insulin action has not been studied. To address this, we used CreLoxP methodology to generate mice with muscle-specific knockout (KO) of GCN5 (mKO), and assessed oral glucose tolerance (OGT) and muscle insulin action (via an ex vivo 2- deoxyglucose uptake [2DOGU] assay in isolated soleus and extensor digitorum longus [EDL] muscles) in mice on a low-fat diet (LFD) or high-fat diet (HFD). GCN5 gene expression and activity in skeletal muscle was decreased 86% and 96%, respectively, in mKO vs. floxed/wildtype (WT) littermates. In LFD mice, there were no genotype differences in OGT or insulin-stimulated 2DOGU. As expected, 3 weeks of HFD impaired OGT in WT mice by ~40%. Interestingly, however, this impairment was exacerbated in mKO mice, which were ~70% worse than LFD-fed mice. These results demonstrate that KO of GCN5 in skeletal muscle does not enhance skeletal muscle insulin sensitivity, and that it may be important for limiting the negative effects of HFD-feeding on glucose tolerance and muscle insulin action.