Ketamine is a widely used general anesthetic and has been reported to demonstrate neurotoxicity and neuroprotection. Investigation into the regulatory mechanism of ketamine on influencing neural development is of importance for a better and safer way of relieving pain. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to detect the critical neural associated gene expression, and flow cytometry to detect the neural differentiation effect. Hence, in the present study the underlying mechanism of ketamine (50 nM) on neural differentiation of the mouse embryonic stem cell (mESC) line 46C was investigated. The results demonstrated that a low dose of ketamine (50 nM) promoted the differentiation of mESCs to neural stem cells (NSCs) and activated mammalian target of rapamycin (mTOR) by upregulating the expression levels of phosphorylated (p)‑mTOR. Furthermore, inhibition of the mTOR signaling pathway by rapamycin or knockdown of mTOR suppressed neural differentiation. A rescue experiment further confirmed that downregulation of mTOR inhibited the promotion of neural differentiation induced by ketamine. Taken together, the present study indicated that a low level of ketamine upregulated p‑mTOR expression levels, promoting neural differentiation.