Objective To investigate the effect of exogenous hydrogen sulfide on Aβ_25-35-induced autophagy in mouse Neuro-2a cells and the underlying mechanism. Methods The Neuro-2a cells were randomly divided into control group, Aβ_25-35 treatment group, Aβ_25-35+NaHS group, Aβ_25-35+3-MA (3-methyl adenine) group, NaHS group and Aβ_25-35+NaHS+LY294002 group. In Aβ_25-35 + NaHS group and Aβ_25-35+3-MA group, NaHS and 3-MA were used for pretreatment for 2 h, which was followed by 24 h co-incubation with Aβ_25-35; in Aβ_25-35+NaHS+LY294002 group, after pretreatment with NaHS, LY294002 was administered for 0.5 h before Aβ_25-35 was given. MTT assay was used to detect the viability of Neuro-2a cells. The expression of the autophagy related proteins including Beclin-1, LC3 and P62 was measured by Western blotting analysis. Immunofluorescence was used to detect the expression and distribution of LC3. The formation of autophagosomes was determined by transmission electron microscopy (TEM). Results (1) Compared with control group, Aβ_25-35 group had significantly declined cell viability (P<0.05), significantly increased expression of Beclin-1 and LC3-Ⅱ and decreased P62 expression (P<0.05), accompanied by increased autophagsomes under fluorescence microscope and electron microscope. (2) Compared with Aβ_25-35 group, the cell viability was significantly increased in Aβ_25-35+3-MA and Aβ_25-35+NaHS groups (P<0.05), and expression of Beclin-1 and LC3-Ⅱ was decreased and expression of P62 was increased (P<0.05), accompanied by reduced autophagosomes. (3) The expression of p-Akt and p-mTOR in Aβ_25-35+NaHS group was significantly higher than that in Aβ_25-35 group (P<0.05); however, the expression of p-Akt and p-mTOR was significantly reduced after given the specific PI3K/Akt pathway inhibitor LY294002 compared with Aβ_25-35+NaHS group (P<0.05). Conclusion The exogenous H₂S can protect against Aβ_25-35-induced cytotoxicity, which is probably related to the activation of PI3K/Akt/mTOR pathways and the inhibition of Aβ_25-35-induced cell autophagy.