Objective To observe the protective effect of smilagenin (SMI) on oxidative stress damage of human neuroblastoma cells (SH-SY5Y) and preliminarily investigate the possible molecular mechanisms. Methods SH-SY5Y cells were pretreated with different concentrations (0.01, 0.1, and 1 μmol/L) of SMI or an equal volume of DMSO for 24 h and then added H2O2 of 600 μmol/L. Morphological changes of SH-SY5Y cells were observed under the inverted microscope. Cell viability was detected by the MTT method. Reactive oxygen species (ROS) was detected by the flow cytometer. The expressions of p-Akt/Akt and HSP70 and the effect of PI3K inhibitor LY294002 on the expression of HSP70 were detected by the Western blotting. Results SMI pretreatment improved the morphology and cell viability of SH-SY5Y cells damaged by H2O2 in a dose dependent manner. The cell viability significantly increased after being treated by SMI of 0.1 and 1 μmol/L (P<0.05, P<0.001). Pretreatment by SMI of 1 μmol/L significantly inhibited the increase of ROS induced by H2O2 (P<0.05). H2O2 induced the expression of p-Akt/Akt and HSP70 and SMI pretreatment further increased expressions of p-Akt/Akt and HSP70 (P<0.01, P<0.05). PI3K inhibitor could block the increase of HSP70 expression (P<0.001). Conclusion SMI can significantly protect SH-SY5Y cells against H2O2-induced oxidative stress damage, which may be relevant to down-regulating the intracellular ROS level and highly activating the PI3K/Akt/HSP70 signaling pathway.