The forkhead protein (FoxO) family plays a crucial role in regulating oxidative stress, cell proliferation, and apoptosis. FoxO6, a member of the FoxO family, helps regulate oxidative stress in gastric cancer and hepatocellular carcinoma. However, it is unclear whether FoxO6 participates in the protective effect of isoflurane preconditioning in liver injury caused by oxidative stress in ischemia. In this study, we explored the role and mechanism of FoxO6 in the protective effect of isoflurane preconditioning during hepatocyte injury caused by oxygen-glucose deprivation (OGD). Cells from the human fetal hepatocyte (LO2) line were incubated with 0%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, or 5% isoflurane for 3 h and then exposed to OGD. Data showed that 3% isoflurane preconditioning inhibited FoxO6 expression, caspase-3 activity, and reactive oxygen species production and promoted cell viability. FoxO6 overexpression abolished the effects of 3% isoflurane preconditioning on caspase-3 activity, reactive oxygen species production, and cell viability in these cells. Moreover, FoxO6 regulated nuclear factor erythroid 2-related factor (Nrf2) expression via c-Myc after 3% isoflurane preconditioning and OGD exposure. Thus, isoflurane preconditioning prevented OGD-induced injury in LO2 cells by modulating FoxO6, c-Myc, and Nrf2 signaling.