Dexmedetomidine (Dex) has been reported to be cardioprotective. Differential expression of miR-208b-3p is associated with myocardial injury. But it is unknown that aberrant expression of miR-208b-3p is implicated in myocardial protection of Dex. Hypoxia/reoxygenation (HR) model was established in H9C2 cells. qRT-PCR was performed to detect expression levels of miR-208b-3p in H9C2 undergoing HR, Dex preconditioning, overexpression of miR-208b-3p or inhibition, and to assess expression of Med13 in H9C2 following knockdown of Med13 mRNA. CCK8 and, flow cytometry and Western blot were conducted respectively to examine viability, apoptosis rate and protein expressions of H9C2 subjected to a variety of treatments. Dex preconditioning reduced expression of miR-208b-3p and apoptosis of H9C2 cells caused by HR, while Dex preconditioning increased viability of H9C2. Dex preconditioning increased expression of Med13, which was reduced after knockdown of Med13 mRNA in H9C2. Overexpression of miR-208b-3p attenuated Dex exerted protective effects of myocardial cells, which was reversed by inhibition of miR-208b-3p. Increased expression of Med13 or/and decreased expression of miR-208b-3p decreased expression levels of Wnt/beta-catenin signaling pathway-related proteins (Wnt3a, Wnt5a and beta-catenin), while knockdown of Med13 mRNA or increased expression of miR-208b-3p increased the expression levels of those proteins. Dex protects H9C2 cells against HR injury through miR-208b-3p/Med13/Wnt/beta-catenin signaling pathway axis.