Hepatic ischemia/reperfusion injury (IRI) is a common cause of morbidity and mortality in liver transplantation settings and involves severe cell death and inflammatory responses. MicroRNA-191 has recently been reported to be abnormally expressed in hepatocellular carcinoma and other liver diseases in the regulation of important cellular processes. However, little is known about its function and molecular mechanism in IRI. Here, we demonstrate that miR-191 is significantly upregulated in a cultured cell line during hypoxia/reperfusion (H/R) and in liver tissue during IRI in mice. The activation of miR-191 under hypoxic conditions is mediated by hypoxia-inducible factor-1α (HIF1α) binding to its promoter region. Global miR-191 KO mice were constructed by CRISPR/Cas9 system, and we found that miR-191 deficiency markedly reduces liver tissue damage, cell inflammatory responses and cell death in a mouse hepatic IRI model. Under the H/R condition, miR-191 overexpression promotes G0/G1 cell cycle arrest and cell apoptosis, but inhibition of miR-191 facilitates cell cycle progression and decreases cell death. Mechanistically, upon induction by hypoxia or ischemia, miR-191 suppresses expression of ZO-1-associated Y-box factor (ZONAB) and its downstream factor Cyclin D1, consequently resulting in cell death and tissue injury. Moreover, the effects of miR-191 on cell cycle arrest and cell apoptosis are abrogated by ZONAB overexpression, and vice versa. Taken together, our results indicate an important role of the HIF1α/miR-191/ZONAB signaling pathway in hepatic IRI and suggest miR-191 as a novel therapeutic target for the treatment of liver IRI.