Modern technological lifestyles promote allergic diseases, especially food allergies. The underlying molecular mechanisms remain to be uncovered. Protein acetylation is one of the most important post-translational modifications, and it is involved in regulating multiple body metabolic processes. This study aimed to clarify the effects of a high-fat diet (HFD) on allergy risk and the underlying mechanisms. Four-week-old male C57BL/6J mice were randomly divided into two groups and fed a normal fat diet (NFD) or HFD for 24 weeks. Then, serum lipids were measured, and skeletal muscle was collected for acetylome analysis. Compared with the findings in the NFD group, HFD-fed mice were obese and hyperlipidemic. Acetylome analysis also revealed 32 differentially expressed proteins between the HFD and NFD groups. Among these, eight acetylated proteins were upregulated in the HFD group. In addition, 13 and 11 proteins were acetylated only in the HFD group and NFD group, respectively. These proteins were mainly involved in regulating energy metabolism and mitochondrial function. This study provides information regarding the underlying molecular mechanisms by which HFD promotes allergy.