Background: Fibronectin type III domain containing 5 (FNDC5) and its protein product Irisin are therapeutic targets for obesity-associated disorders. Irisin plays an important role in energy regulation, inducing browning of white adipocytes, and improving obesity. We aimed to investigate the association between muscle Irisin expression and dietary quality. Methods: Twenty-eight female mice were divided into four groups and fed the following experimental diets for 60 days: standard diet (SD), high-carbohydrate diet (HCD), high-fat diet (HFD), and high-protein diet (HPD). We evaluated body weight, food intake, serum total cholesterol, triacylglycerol, and glucose. We also performed glucose tolerance and insulin sensitivity tests. Expression of FNDC5 was evaluated by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of soleus muscle. Western blot was used to assess Irisin protein expression. Results: The major finding of the present study was that HFD and HCD were associated with a downregulation of FNDC5. In addition to these results, we noted a significant reduction in skeletal muscle Irisin level. HPD prevented reductions of both FNDC5 and Irisin levels, as well as increased brown adipose tissue, compared to the control group. Conclusions: In conclusion, we observed that the HPD type of diet can change both FNDC5 expression and Irisin levels. Thus, the HPD might be the most appropriate diet to achieve high amounts of Irisin, a target molecule for the treatment of obesity and its co-morbidities.