AIM: To investigate the protective effect of midazolam (MID) on cardiac function and angiogenesis in myocardial infarction (MI) rats and its effect on c-Jun N-terminal kinase-signal transducer and activator of transcription 3 (JNK/STAT3) pathway. METHODS: MI rat model was established by left anterior descending coronary artery ligation method. A total of 68 rats were modeled. After excluding the rats that died (n=3, mortality rate≈4.4%) or failed modeling (n=5), the 60 MI rats included in this experiment were randomly divided into the model group (Model group), MID low, medium and high dose group (1 mg/kg MID-L group, 3 mg/kg MID-M group, 6 mg/kg MID-H group), MID high dose+JNK activator Anisomycin group (6 mg/kg MID-H+Anisomycin group), 12 rats in each group. Another 12 rats undergoing sham surgery were selected as the control group. All rats were echocardiographic and evaluated for cardiac function. The myocardial tissue pathological morphology and the myocardial fibrosis degree were observed by hematoxylin-eosin (HE) staining and Masson staining. The serum myocardial injury markers ［creatine kinase isoenzyme (CK-MB) and brain natriuretic peptide (BNP)］ and inflammatory factors ［tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and C-reactive protein (CRP)］, myocardial tissue oxidative stress indicators ［malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH)］ levels were detected by enzyme-linked immunosorbent assay (ELISA). The vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), p-JNK, JNK, p-STAT3 and STAT3 proteins expression levels were detected by Protein blot method (Western blot). RESULTS: Compared with Control group, the myocardial tissue damage degree in Model group was more severe, the myocardial interstitial fibrosis degree was greatly deepened, the collagen volume fraction was significantly increased, the left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), CK-MB, BNP, CRP, TNF-α, IL-1β and MDA levels were significantly increased, the left ventricular ejection fraction (LVEF), left ventricular short axis shortening rate (LVFS), GSH and SOD levels were significantly decreased, and the VEGF, VEGFR2 proteins expression levels were significantly decreased, the p-JNK/JNK and p-STAT3/STAT3 were significantly increased (P<0.05). Compared with Model group, with the MID dose increase, the myocardial tissue damage degree in MID-L, MID-M and MID-H groups was significantly reduced, the myocardial interstitial fibrosis degree was reduced, the collagen volume fraction was decreased, the LVESD, LVEDD, CK-MB, BNP, CRP, TNF-α, IL-1β and MDA levels were significantly decreased, the LVEF, LVFS, GSH and SOD levels were significantly increased, and the VEGF, VEGF2R protein expression levels were significantly increased, p-JNK/JNK and p-STAT3/STAT3 were significantly decreased (P<0.05). Compared with MID-H group, the myocardial tissue damage degree in MID-H+Anisomycin group was more severe, the myocardial interstitial fibrosis degree was greatly deepened, the collagen volume fraction was significantly increased, the cardiac function was weakened, the oxidative stress and inflammation were aggravated, LVESD, LVEDD, CK-MB, BNP, CRP, TNF-α, IL-1β and MDA levels were increased, the LVEF, LVFS, GSH, SOD, VEGF and VEGFR2 proteins expression levels were significantly decreased, p-JNK/JNK and p-STAT3/STAT3 were significantly increased (P<0.05). CONCLUSION: MID may improve myocardial tissue damage in MI rats by regulating JNK/STAT3 pathway, reduce oxidative stress and inflammatory response, promote angiogenesis, so as to play a role in cardiac protection.