Aim To investigate the effect of antagomiR-21 on endothelium-dependent relaxation of coronary artery in type 2 diabetes mellitus (T2DM) rats by regulating silent information regulator 1 (SIRT1) and its mechanism. Methods T2DM rat model was established by intraperitoneal injection of streptozotocin and fed with high-fat diet. 28 adult rats were randomly divided into model group, antagomiR-NC group, antagomiR-21 group, and antagomiR-21＋SIRT1 inhibitor EX527 group, with 7 rats in each group; in addition, 7 normal rats fed with common diet were used as control group. The changes of coronary artery flow in rats were observed, and the diastolic effect of coronary artery in rats was observed by isolated vascular ring perfusion technique. Human coronary artery endothelial cells (HCAEC) were divided into mannitol group, high glucose group, high glucose＋antagomiR-NC group, high glucose＋antagomiR-21 group, high glucose＋antagomiR-21＋EX527 group. The expression of miR-21 and SIRT1 mRNA was detected by qRT-PCR, and the expression of SIRT1, phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), endothelial nitric oxide synthase (eNOS) and its phosphorylated protein was detected by Western blot. Results Compared with the control group, the levels of miR-21 in the coronary arteries of T2DM model rats increased by 96.88%, while the expression levels of SIRT1 protein and mRNA, and coronary artery flow decreased by 40.85%, 64.29% and 22.15%, respectively (P＜0.05); compared with the mannitol group, in vitro high glucose treatment caused an increase of 285.71% in miR-21 expression in HCAEC, while the expression levels of SIRT1 protein and mRNA decreased by 44.78% and 74.51%, respectively (P＜0.05). The intervention of antagomiR-21 resulted in a decrease of 77.42% and 58.66% in both in vivo and in vitro miR-21 levels, an increase of 55.56% and 91.43% in SIRT1 protein levels, and an increase of 88.57% and 97.30% in SIRT1 mRNA levels, respectively (P＜0.05). Compared with the model group, the intervention of antagomiR-21 resulted in a 19.23% increase in coronary artery flow in rats, and a 111.89%, 41.88%, 41.98%, and 30.01% increase in coronary artery relaxation rate induced by 10－8 mol/L, 10－7 mol/L, 10－6 mol/L and 10－5 mol/L acetylcholine (Ach), respectively (P＜0.05), and the coronary artery contraction rate in rats decreased by 36.71%, 47.90%, 49.19% and 45.27% induced by 10－8 mol/L, 10－7 mol/L, 10－6 mol/L and 10－5 mol/L phenylephrine (Phe) (P＜0.05). After the intervention of antagomiR-21, the phosphorylation levels of PI3K, Akt and eNOS in HCAEC increased by 48.48%, 81.40% and 134.29%, respectively, compared to the high glucose group (P＜0.05). EX527 treatment can significantly reverse the above changes caused by antagomiR-21 in vitro and in vivo (P＜0.05). Conclusion AntagoniR-21 can activate the PI3K/Akt/eNOS signaling pathway by upregulating SIRT1 expression, thereby improving endothelium-dependent relaxation of coronary artery in T2DM rats.