Autophagy is an essential cytoprotective response against pathologic stresses that selectively degrades damaged cellular components. Impaired autophagy contributes to organ injury in multiple diseases, including ischemia/reperfusion (I/R), but the exact mechanism by which impaired autophagy is regulated remains unclear. Several researchers have demonstrated that microRNAs (miRNAs) negatively regulate autophagy by targeting autophagy-related genes (ATGs). Therefore, the effect of ATG-related miRNAs on I/R remains a promising research avenue. In our study, we found that autophagy flux is impaired during intestinal I/R. A miRNA microarray analysis showed that miR-665-3p was highly expressed in the I/R group, which was confirmed by qRT-PCR. Then, we predicted and proved that miR-665-3p negatively regulates ATG4B expression in Caco-2 and IEC-6 cells. In ileum biopsy samples from patients with intestinal infarction, there was an inverse correlation between miR-665-3p and ATG4B expression, which supports the in vitro findings. Moreover, based on miR-665-3p regulation of autophagy in response to hypoxia/reoxygenation in vitro, gain-of-function and loss-of-function approaches were used to investigate the therapeutic potential of miR-665-3p. Additionally, we provide evidence that ATG4B is indispensable for protection upon inhibition of miR-665-3p. Finally, we observed that locked nucleic acid-modified inhibition of miR-665-3p in vivo alleviates I/R-induced systemic inflammation and apoptosis via recovery of autophagic flux. Our study highlights miR-665-3p as a novel small molecule that regulates autophagy by targeting ATG4B, suggesting that miR-665-3p inhibition may be a potential therapeutic approach against inflammation and apoptosis for the clinical treatment of intestinal I/R.