Long-term exposure to isoflurane may induce long-term developmental neurotoxicity and cognitive impairments in the neonatal brains. Trilobatin, a leaf extract from the Chinese traditional sweet tea Lithocarpus polystachyus Rehd, possesses various biological properties including anti-inflammatory and anti-oxidant properties. Our study aimed to explore the neuroprotective effect of trilobatin on isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells. The effects of trilobatin on cell viability, LDH release, apoptosis, and caspase-3/7 activity in isoflurane-induced HT22 cells were explored by CCK-8, LDH release assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. Oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and malonyldialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT). The expression of nuclear erythroid-2 related factor 2 (Nrf2), nuclear Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) was determined by western blot and qRT-PCR. Results suggested that exposure to isoflurane significantly reduced cell viability and increased LDH release, apoptotic rate and caspase-3/7 activity in HT22 cells, which were abolished by trilobatin. Trilobatin reversed isoflurane-induced increase of ROS and MDA levels and reduction of SOD and CAT activities in HT22 cells. Additionally, trilobatin promoted the nuclear translocation of Nrf2 as well as the mRNA and protein expression of HO-1 and NQO1 in HT22 cells exposed to isoflurane. Nrf2 knockdown attenuated the effects of trilobatin on isoflurane-induced viability reduction, LDH release, apoptosis, and oxidative stress in HT22 cells. Overall, trilobatin protected HT22 cells against isoflurane-induced neurotoxicity via activating the Nrf2/antioxidant response element (ARE) pathway.