Arecoline, the most abundant alkaloid of the areca nut, induces toxicity to neurons. Hydrogen sulfide (H2S) is an endogenous gas with neuroprotective effects. We recently found that arecoline reduced endogenous H2S content in PC12 cells. In addition, exogenously administration of H2S alleviated the neurotoxicity of arecoline on PC12 cells. Increasing evidence has demonstrated the neuroprotective role of improvement of autophagic flux. Therefore, the aim of the present work is to explore whether improvement of autophagic flux mediates the protection of H2S against arecoline-caused neurotoxicity. Transmission electron microscope (TEM) for observation of ultrastructural morphology. Western blotting was used to detect protein expression of the related markers. Functional analysis contained LDH release assay, Hoechst 33,258 nuclear staining and flow cytometry were used to detect cytotoxicity and apoptosis. In the present work, we found that arecoline disrupted autophagy flux in PC12 cells as evidenced by accumulation of autophagic vacuoles, increase in LC3II/LC3I, and upregulation of p62 expression in PC12 cells. Notably, we found that sodium hydrosulfide (NaHS), the donor of H2S improved arecoline-blocked autophagy flux in PC12 cells. Furthermore, we found that blocking autophagic flux by chloroquine (CQ), the inhibitor of autophagy flux, antagonized the inhibitory role of NaHS in arecoline-induced cytotoxicity apoptosis and endoplasmic reticulum (ER) stress. In conclusion, H2S improves arecoline-caused disruption of autophagic flux to exert its protection against the neurotoxicity of arecoline.