Biatractylolide, isolated from the ethyl acetate extract of Atractylodes macrocephala, has shown various pharmacological activities such as antitumor and antioxidant activities. In this work, we aim to study the protective effect of biatractylolide on glutamate-induced rat adrenal pheochromocytoma cell (PC12) and human bone marrow neuroblastoma cell line (SH-SY5Y) injury and preliminarily explore its mechanism. The results showed that glutamate was cytotoxic with an inhibitory concentration 50% (IC50) of 8.5 mM in PC12 and 10 mM in SH-SY5Y cells. In this work, the preincubation with biatractylolide (10, 15, and 20 μM) observably improved cell viability, inhibited the apoptosis of cells induced by glutamate, and reduced the activity of LDH. AO staining revealed that apoptosis of cells was decreased. Additionally, the results of western blotting manifested that pretreatment with biatractylolide could downregulate GSK3β protein expression and upregulate p-Akt protein expression, thereby protecting PC12 and SH-SY5Y cells from injury. All these findings indicate that biatractylolide has a neuroprotective effect on glutamate-induced injury in PC12 and SH-SY5Y cells through a mechanism of the PI3K-Akt-GSK3β-dependent pathways.