Abstract Objective To explore the role and potential mechanism of glutamate in enhancing insulin sensitivity by aerobic exercise. This research may provide a novel strategy for preventing metabolic diseases thorugh precise exercise interventions. Methods To investigate the effects of elevated circulating glutamate on insulin sensitivity and its potential mechanisms, 18 male C57BL/6 mice aged 6 to 8 weeks were randomly divided into 3 groups: a control group (C), a group receiving 500 mg/kg glutamate supplementation (M), and a group receiving 1 000 mg/kg glutamate supplementation (H). The intervention lasted for 12 weeks, with treatments administered 6 d per week. Following the intervention, an insulin tolerance test (ITT) and a glucose tolerance test (GTT) were conducted. Circulating glutamate levels were measured using a commercial kit, and the activity of the skeletal muscle InsR/IRS1/PI3K/AKT signaling pathway was analyzed via Western blot. To further investigate the role of glutamate in enhancing insulin sensitivity through aerobic exercise, 30 male C57BL/6 mice were randomly assigned to 3 groups: a control group (CS), an exercise intervention group (ES), and an exercise group combined with glutamate supplementation (EG). The ES group underwent treadmill-based aerobic exercise, while the EG group received glutamate supplementation at a dosage of 1 000 mg/kg in addition to aerobic exercise. The intervention lasted for 10 weeks, with sessions occurring 6 d per week, and the same procedures were followed afterward. To further elucidate the mechanism by which glutamate modulates the InsR/IRS1/PI3K/AKT signaling pathway, C2C12 myotubes were initially subjected to graded glutamate treatment (0, 0.5, 1, 3, 5, 10 mmol/L) to determine the optimal concentration for cellular intervention. Subsequently, the cells were divided into three groups: a control group (C), a glutamate intervention group (G), and a glutamate combined with MK801 (an NMDA receptor antagonist) intervention group (GK). The G group was treated with 5 mmol/L glutamate, while the GK group received 50 μmol/L MK801 in addition to 5 mmol/L glutamate. After 24 h of intervention, the activity of the InsR/IRS1/PI3K/AKT signaling pathway was analyzed using Western blot. Results Compared to the mice in group C, the circulating glutamate levels, the area under curve (AUC) of ITT, and the AUC of GTT in the mice of group H were significantly increased. Additionally, the expression of p-InsR β, IRS-1, p-Akt, and p-mTOR proteins in skeletal muscle was significantly downregulated. Compared to the mice in group CS, the circulating glutamate levels, the AUC of ITT, and the AUC of GTT in the mice of group ES were significantly reduced. Additionally, the expression levels of p-InsR β, IRS-1, p-Akt, and p-mTOR proteins in skeletal muscle was significantly upregulated. There were no significant changes observed in the mice of group EG. Compared to the cell in group 0 mmol/L, the expression levels of p-InsR β, p-IRS-1, p-PI3K, and p-AKT proteins in group 5 mmol/L were significantly downregulated. Compared to the cells in group C, the expression levels of p-InsR β, p-IRS-1, p-PI3K, and p-AKT proteins were significantly downregulated. No significant changes were observed in the cells of group GK. Conclusion Long-term aerobic exercise can improve insulin sensitivity by lowering circulating levels of glutamate. This effect may be associated with the upregulation of the InsR/IRS1/Akt signaling pathway avtivity in skeletal muscle. Furthermore, glutamate can weaken the activity of the InsR/IRS1/PI3K/Akt signaling pathway in skeletal muscle, potentially by binding to NMDAR expressed in skeletal muscle.