Reperfusion may cause injuries to the myocardium in ischemia situation, which is called ischemia/reperfusion (I/R) injury. The study aimed to explore the roles of microRNA-29b (miR-29b) in myocardial I/R injury. Myocardial I/R injury rat model was established. Differentially expressed miRNAs between the model rats and the sham-operated rats were analyzed. miR-29b expression in myocardial tissues was measured. Gain-of-function of miR-29b was performed, and then the morphological changes, infarct size, myocardial function, oxidative stress, and the cell apoptosis in myocardial tissues were detected. The target relation between miR-29b and PTEN was detected through bio-information prediction and dual luciferase reporter gene assay. Activation of Akt/eNOS signaling was detected. H9C2 cells were subjected to hypoxia/reoxygenation treatment to perform in vitro experiments. I/R rats presented severe inflammatory infiltration, increased infarct size and cell apoptosis, increased oxidative stress and decreased myocardial function. miR-29b was downregulated in I/R rats, and up-regulation of miR-29b reversed the above changes. miR-29b directly bound to PTEN, and overexpression of miR-29b reduced PTEN expression level and increased the protein levels of p-Akt/Akt and p-eNOS/eNOS. In vivo results were confirmed in in vitro experiments. This study provided evidence that miR-29b could alleviate the myocardial I/R injury in vivo and in vitro by inhibiting PTEN expression and activating the Akt/eNOS signaling pathway.© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.