Exosomes have been shown to effectively regulate the biological functions of target cells. Here, we investigated the protective effect and mechanism of hypoxia-induced renal tubular epithelial cells (TECs)-derived exosomes on acute tubular injury. We found that in vitro hypoxia-induced tubular exosomes (Hy-EXOs) were protective in acute tubular injury by promoting TECs proliferation and improving mitochondrial functions. By using exosome miRNA sequencing, we identified miR-20a-5p was abundant and was a key mechanism for the protective effect of Hy-EXOs on tubular injury as up-regulation of miR-20a-5p enhanced but down-regulation of miR-20a-5p inhibited the protective effect of Hy-EXOs on tubular injury under hypoxia conditions. Further study in a mouse model of ischemia–reperfusion-induced acute kidney injury (IRI-AKI) also confirmed this notion as pre-treating mice with the miR-20a-5p agomir 48 h prior to AKI induction was capable of inhibiting IRI-AKI by lowering serum levels of creatinine and urea nitrogen, and attenuating the severity of tubular necrosis, F4/80(+) macrophages infiltration and vascular rarefaction. Mechanistically, the protective effect of miR-20a-5p on acute kidney injury (AKI) was associated with inhibition of TECs mitochondrial injury and apoptosis in vitro and in vivo. In conclusion, miR-20a-5p is enriched in hypoxia-derived tubular exosomes and protects against acute tubular injury. Results from the present study also reveal that miR-20a-5p may represent as a novel therapy for AKI.