Objective To investigate the effects and molecular mechanisms of midazolam (MID) on myocardial cell injury induced by hypoxia. Methods To determine the optimal MID concentration, H9C2 cells were pretreated with different concentrations of MID for 24 h, induced with hypoxia, and the survival rate was assayed using the cell counting kit (CCK-8). H9C2 cells were divided into seven groups: normal control ( NC); hypoxia; MID + hypoxia; microRNA (miRNA) negative control (NC) + hypoxia; miR-290-5p + hypoxia; anti-miR-NC + MID + hypoxia; and anti-miR-290- 5p + MID + hypoxia. Flow cytometry was used to detect apoptosis. Real-time quantitative PCR (RT-qPCR) was used to detect miR-290-5p expression. Western blot was used to detect phosphatidylinositol-3-kinase / protein kinase B ( PI3K/ AKT) signaling pathway-related protein expression. Results The cell survival rate of H9C2 cells pretreated with MID at 8, 16 and 32 μmol / L was significantly increased after hypoxia (P < 0. 05); 16 μmol / L was the optimal concentration. Compared with the NC group, the apoptosis rate of H9C2 cells in the hypoxia group was significantly increased, while the expression of miR-290-5p and the PI3K pathway activity were significantly reduced ( P< 0. 05). Compared with the hypoxia group, the apoptosis rate of H9C2 cells in the MID + hypoxia group was significantly reduced, and the expression of miR-290-5p and the activity of the PI3K pathway were significantly increased (P< 0. 05). Compared with the MID + hypoxia group, the survival rate of H9C2 cells in the miR-290-5p + hypoxia group was significantly increased, and the apoptosis rate was significantly reduced (P< 0. 05). Compared with the anti-miR-NC + MID + hypoxia group, the survival rate of H9C2 cells in the anti-miR-290-5p + MID + hypoxia group was significantly reduced, the apoptosis rate was significantly increased, and PI3K pathway activity was significantly reduced (P<0.05). Conclusions Midazolam protected hypoxia-induced cardiomyocyte damage by upregulating miR-290-5p to activate PI3K/ AKT signaling pathway.