Objective To investigate the protective effect of hesperidin extract from orange peel on oxidative stress in an in vitro model of nonalcoholic fatty liver disease (NAFLD). Methods CCK-8 method was used to screen the drug concentrations of oleic acid (OA) and hesperidin (HES). An in vitro NAFLD model was established by OA action on LO2 cells, with a normal control group (NC group), model group (Model group), low-dose group (L-HES), medium- dose group (M-HES) and high-dose group (H-HES group). The groups were treated with HES for 24 h; oil red O staining was performed to detect intracellular ROS, MDA, SOD and CAT, and Western blot was used to detect the expression of Nrf2, HO-1, NQO1 and GCLC proteins in LO2 cells. Results Compared with result in the NC group, numerous intracellular lipid droplets were formed in the Model group (P<0.05); ROS and MDA expression increased, SOD and CAT expression decreased (P< 0.05) and Nrf2, HO-1, NQO1 and GCLC expression were all down-regulated ( P< 0.05). Compared with observations the Model group, fewer lipid droplets were observed in the HES low-medium-high group (P< 0.05). ROS and MDA production decreased, SOD and CAT contents increased (P<0.05) and Nrf2, HO-1, NQO1 and GCLC protein expressions were all upregulated (P<0. 05). Conclusions HES has a protective effect on the NAFLD in vitro model through anti-oxidative stress, which may be related to the activation of Nrf2 / HO-1 signaling pathway.