Methionine restriction (MR) is an effective dietary strategy to regulate energy metabolism and alleviate oxidative stress and inflammation in the body, especially in the middle-aged and elderly population. However, the high methionine content of meat products makes this dietary strategy impossible to combine with protein supplementation and MR. Highland barley (HB), a low-methionine cereal, not only provides the body with protein but also has improved glucose metabolism and antioxidant and anti-inflammatory properties. Therefore, this study evaluated the feasibility of HB as a source of methionine-restricted dietary protein and the potential mechanisms. Middle-aged C57BL/6J mice were fed a control diet (CON), a high-fat diet (HFD), a whole-grain HB high-fat diet (HBHF), or a HBHF + methionine diet (HBHFmet) for 25 weeks. The results showed that the HBHF could keep the body weight, fasting glucose, insulin, homeostasis model assessment of insulin resistance (HOMA-IR), blood lipids, inflammation, and oxidative stress of HFD mice at normal levels. Compared with the HFD groups, HBHF inhibited pancreatic cell apoptosis and improved insulin secretion while improving hepatic and skeletal muscle glucose metabolism. However, these efficacies were attenuated in HBHFmet group mice. These findings suggest that HBHF has an MR strategy.