Propofol, a general intravenous anesthetic, has been demonstrated to cause a profound neuroapoptosis in the developing brain followed by long-term neurocognitive impairment. Our study aimed to examine the neuroprotective effect of neuronal PAS domain protein 4 (NPAS4), an activity-dependent neuron-specific transcription factor, on propofol-induced neurotoxicity in hippocampal neuronal HT22 cells. The differentially expressed genes in HT22 cells after treatment with propofol were screened from Gene Expression Omnibus dataset GSE106799. NPAS4 expression in HT22 cells treated with different doses of propofol was investigated by qRT-PCR and Western blot analysis. Cell viability, lactate dehydrogenase (LDH) release, caspase-3 activity, and apoptosis were evaluated by MTT, a LDH-Cytotoxicity Assay Kit, a Caspase-3 Colorimetric Assay Kit, and TUNEL assay, respectively. The protein levels of LC3-I, LC3-II, Beclin 1, p62 and NPAS4 were detected using Western blot analysis. Propofol treatment concentration-dependently decreased NPAS4 expression in HT22 cells. Propofol treatment inhibited cell viability, increased LDH release and caspase-3 activity, and induced apoptosis and autophagy in HT22 cells. NPAS4 overexpression suppressed propofol-induced cell injury and autophagy in HT22 cells. Mechanistically, autophagy agonist rapamycin attenuated the neuroprotective effect of NPAS4 in propofol-treated HT22 cells. In conclusion, NAPS4 overexpression protected hippocampal neuronal HT22 cells against propofol-induced neurotoxicity by reducing autophagy.