Objective To explore the role of alanyl-glutamine (Ala-Gln) in myocardial infarction and its possible mechanism. Methods Thirty-six SPF male Wistar rats were randomized into four groups (n=9 each):sham operation group, model group (myocardial ischemic group), treatment group treated with isoproterenol [ISO, 5mg/(kg·d)]+Ala-Gln [0.75mg/(kg·d)], and quercetin group treated with ISO[5mg/(kg·d)]+quercetin [100mg/(kg·d)] +Ala-Gln [0.75mg/(kg·d)]+dimethylsulfoxide. After successful modeling, the treatment and quercetin groups were treated for 7 days,while the sham operation group were treated with the same amount of saline. The rats were sacrificed after 7 days. HE staining was used to observe myocardial ischemic necrosis, area of myocardial ischemic necrosis was calculated by direct weighing; immunohistochemical method was used to observe the expression and distribution of heat shock protein 70 (HSP70) and semi-quantitative analysis was performed; and chemical colorimetric method was used to detect the activities of serum myocardial creatases (aspartate aminotransferase, AST; lactate dehydrogenase, LDH; creatine kinase, CK) and malonaldehyde (MDA), and the level of superoxide dismutase (SOD). SPSS statistics 13.0 was used for data analysis. Results The percentage of myocardial infarction mass in model group [(0.241 ± 0.054)%], treatment group [(0.165 ± 0.372)]% and quercetin group [(0.248 ± 0.035)%] was statistically significantly different with that of the sham operation group [(0.01 ± 0.002)%,P＜0.01], and the mass of myocardial infarction in the treatment group was significantly smaller than that in the model group and quercetin group (P＜0.01). There was no significant difference in the expression levels of HSP70 between the sham operation group (0.154 ± 0.036), model group (0.151 ± 0.033) and quercetin group [(0.151 ± 0.025), P>0.05], but that of the treatment group (0.286 ± 0.056) was significantly higher than those of the other groups (P＜0.01). Compared with the sham operation group, CK, AST and LDH increased in model group, treatment group and quercetin group (P＜0.01), and they were lower in the treatment group than in the model group and the quercetin group (P＜0.01). Compared with the sham operation group, MDA in the model group and quercetin group increased (P＜0.05), and SOD decreased (P＜0.05). MDA was lower and SOD was higher (P＜0.05 for both) in the treatment group than that in the model group and quercetin group. Conclusion Ala-Gln has a protective effect on myocardial infarction in rats. Its mechanism may be related to the induction of HSP70 expression, reduction of MDA activity and increase of SOD level, thus enhancing the antioxidant capacity of cardiomyocytes and reducing the damage of active free radicals.