Objective To investigate the protective effect of dexmedetomidine preconditioning on ischemia- reperfusion injury ( MIRI) in isolated rat hearts and its potential regulatory mechanism via the connexin 43 ( Cx43) / mitochondrial ATP-sensitive potassium channel ( mito-KATP) signaling axis. Methods The isolated heart Langendorff perfusion model was used. The random number table method was used to divide 50 isolated hearts into 5 groups (n= 10 / group): control ( Blank group ), ischemia-reperfusion ( I/ R group ), ischemia-reperfusion + dexmedetomidine preconditioning (I/ R+Dex group), ischemia-reperfusion+dexmedetomidine preconditioning+mito-KATP channel blocker 5- HD (I/ R+Dex+5-HD group), and ischemia-reperfusion+mito-KATP channel blocker (I/ R+5-HD group) groups. The rat model of isolated myocardial ischemia-reperfusion injury was prepared by stopping perfusion for 30 min and reperfusion for 120 min. The Blank group was continuously perfused with K-Hsolution for 180 min. The I/ R group was perfused with KH solution for 30 min, stopped for 30 min, and then perfused with KH solution for 120 min, causing MIRI injury. The I/ R+ Dex group was perfused with KH solution containing 10 mg / L dexmedetomidine for 30 min , stopped for 30 min, and then perfused with KH solution for 120 min. The I/ R+Dex+5-HD group was perfused with KH solution containing 10 mg / L of mito-KATP channel blocker 5-hydroxykulanic acid (5-HD) for 15 min, perfused with KH solution containing 10 mg / L of dexmedetomidine for 15 min, stopped for 30 min, and then perfused with KH solution for 120 min. The I/ R+5-HD group was perfused with KH solution containing 10 mg / L of 5-HD for 30 min, stopped for 30 min, and then perfused with KH solution for 120 min. Triphenyltetrazolium chloride staining was used to detect the proportion of cardiac infarction in each group. The expression of Cx43 in rat hearts from each group was detected by immunohistochemistry. Western blot was used to detect the heart expression levels of p-Cx43 in each group. Results Compared with the Blank group, the myocardial infarction area of the I/ R, I/ R +Dex, I/ R +Dex + 5-HD and I/ R + 5-HD groups were significantly increased, and the expression levels of Cx43 and p-Cx43 were significantly decreased. Compared with the I/ R group, the area of myocardial infarction in the I/ R+Dex and I/ R+Dex+5-HD groups were significantly reduced, and the expression levels of Cx43 and p- Cx43 were increased. Compared with the I/ R group, the area of myocardial infarction in the I/ R + 5-HD group was significantly increased, and the expression levels of Cx43 and p-Cx43 were significantly reduced.Compared with the I/ R+ Dex group, the areas of myocardial infarction in the I/ R+Dex+5-HD and I/ R+5-HD groups,were significantly reduced, and the expressions of Cx43 and p-Cx43 were reduced. The differences were statistically significant ( P< 0.05 ). Conclusions Dexmedetomidine preconditioning can promote the expression and phosphorylation of Cx43, and the opening of the mito-KATP channel, and reduce ischemia-reperfusion injury of isolated hearts.