Objective The imbalance of glucagon secretion plays an important role in the development of diabetes. The newly discovered ACE2/Ang(1-7)/Mas pathway is an vital branch of the RAS system and has regulatory effects on islet function, but its effect on glucagon secretion is still unknown. The article aimed to investigate the effect and possible mechanisms of AVE0991, a Mas receptor agonist on glucagon secretion of diabetic db/db mice. Methods A tolal of 30 db/db mice were randomized to AVE group and model group (n=15), and their age-matched nondiabetic db/m mice were selected as the normal group (n=15). The mice in AVE group were treated with AVE0991 20mg/kg/d and those in model group were treated with placebo via gavage for 6 weeks. The metabolic indicators were measured every week. After 6 weeks of treatment, intraperitoneal glucose tolerance test (IPGTT) and islet perifusion were performed to evaluate glucagon release kinetics in vivo and vitro. Double-label immunofluoresence assay was adoppted to assess the content of α and β cells. Moreover, qRT-PCR and western blot were employed to detect the GCK expression in islets. Results The fasting blood sugar[(19.1±0.8)mmol/L] and glucose tolerance [(14.1±0.5) mU/L]of AVE group were significantly lower than those of the model group[(25.3±3.0)mmol/L,(17.3±1.8)mU/L](P＜0.05) and still higher than those of normal group[(6.3±0.5)mmol/L,(5.7±0.3)mU/L](P＜0.05). At the 30, 60, and 120 min after IPGTT, the blood glucose level and glucagon level in AVE group were lower than model group, but still higher than the normal group (P<0.05). During low glucose perfusion, the glucagon secretion level of the islets of normal group [(20.6±0.5 pmol/L)] was lower than that of model group [(29.1±0.7) pmol/L)] and AVE group [(27.6±0.8) Pmol/L], and the difference was statistically significant (P<0.05). During high glucose perfusion, there was a statistically significant difference in glucagon level between AVE group and model group at 30 and 60 min(P<0.05). Semi-quantitative analysis showed that the islet α-cell content of model group [(3.3±0.7) mg] was significantly higher than that of normal group [(1.2±0.3) mg] (P<0.05), and the β cell content [(2.4±0.6) mg] was significantly lower than that of normal group [(4.8±0.3) mg] (P<0.05); While compared with the model group, the islet α-cell content [(1.8±0.4) mg] decreased significantly (P<0.05) and the β-cell content [(4.2±0.5) mg] increased significantly in AVE group (P<0.05). The expression levels of GCK mRNA and protein in model group were lower than those in normal group (P<0.05). Conclusion Activation of Mas receptors can improve glucose metabolism by reducing the secretion of glucagon after glucose load in db/db diabetic mice. The mechanism may be related to the decrease of islet α cell content and the increase of islet GCK expression.