The aim of this study was to investigate the effects of dexmedetomidine (Dex) on hepatic ischemia/reperfusion injury (HIRI) and the underlying mechanism. The in vitro HIRI was induced by culturing HL-7702 cells, a human hepatocyte cell line, under 24 h of hypoxia and 12 h of reoxygenation. Quantitative real time PCR (qRT-PCR) and Western blot were performed to detect the expression levels of long non-coding RNA MALAT1, microRNA-126-5p (miR-126-5p) and high mobility group box-1 (HMGB1). Bioinformatics prediction and double luciferase assay were used to verify the targeting relationship between miR-126-5p and MALAT1, HMGB1. Reactive oxygen species (ROS), malondialdehyde (MDA) and ATP levels in culture medium were detected by corresponding kits. The results showed that Dex significantly reduced the levels of ROS and MDA, but increased the level of ATP in HL-7702 cells with HIRI. HIRI up-regulated the expression levels of MALAT1 and HMGB1, and down-regulated the level of miR-126-5p. Dex reversed these effects of HIRI. Furthermore, Dex inhibited HIRI-induced cellular apoptosis, whereas MALAT1 reversed the effect of Dex. This inhibitory effect of Dex could be restored by up-regulation of miR-126-5p. The results suggest that Dex protects hepatocytes from HIRI via regulating MALAT1/miR-126-5p/HMGB1 axis.