Autophagy is an important self-adaptive mechanism that is involved in inhibiting reactive oxygen species (ROS) in spinal cord neurons. Pterostilbene, a natural plant extract, has been demonstrated to possess antioxidant effects; however, it has not yet been investigated whether pterostilbene could activate autophagy and protect spinal cord neurons from oxidative stress. In the present study, primary spinal cord neurons of Sprague Dawley rats were cultured. Cell counting kit-8 analysis was used to detect cytotoxicity of pterostilbene. Cells were treated with various doses of pterostilbene for 24 and 48 h, respectively, and H2O2 was used to induce ROS production. Western blot analysis was performed to assess the protein expression of microtubule-associated protein 1 light chain 3 (LC3)-II, Beclin-1, p62, p-p70S6K and p-mechanistic target of rapamycin (mTOR). Furthermore, the green fluorescent protein (GFP)-LC3 assay was used to detect the level of autophagy level and activation mechanism. 2,7-Dichlorofluorescin diacetate and MitoSOX Red staining were used to detect ROS production, and Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling assay was used to analyze apoptosis percentage. ATG5 small interfering (si)RNA transfection was used to analyze the involvement of autophagy. A dose-dependent increase in the expression of LC3-II and Beclin-1, as well as the p62 decline, were observed in the pterostilbene-treated neurons; however, p-p70S6K and p-mTOR expression was inhibited by pterostilbene. Pterostilbene increased the expression of LC3-II in H2O2-treated cells, and GFP-LC3 analysis demonstrated an increased number of autophagosomes. Furthermore, pterostilbene significantly inhibited the ROS production and apoptosis induced by H2O2; however, ATG5 siRNA transfection significantly reversed the protection of pterostilbene. These results indicate that pterostilbene may inhibit the ROS production and apoptosis in spinal cord neurons by activating autophagy via the mTOR signaling pathway.