Objective To investigate the protective effect of Schisandrin B (Sch B) against high glucose (HG)-induced cytotoxicity in rat adrenal pheochromocytoma (PC12) cells and elucidate its underlying mechanisms. Methods MTT assay was adopted to screen the optimal concentration and treatment duration of HG for inducing PC12 cell injury. PC12 cells were treated with different concentrations of Sch B to screen non-cytotoxic drug concentrations. An in vitro model of diabetic cognitive dysfunction was established by HG stimulation. After Sch B intervention, cell viability, cell apoptosis and intracellular reactive oxygen species (ROS) levels were detected. The release of lactate dehydrogenase (LDH) in culture medium was determined by commercial kits to evaluate the degree of cell injury. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in cells were measured to assess the level of oxidative stress. Results This study confirmed that treatment of PC12 cells with 125 mmol·L^-1 glucose for 48 h could establish a stable HG-induced cell injury model. Compared with the normal control group, 48 h of HG exposure markedly reduced PC12 cell viability (P<0.01), while the apoptosis rate, LDH release and intracellular oxidative stress level were significantly elevated. Sch B at concentrations ranging from 1 to 20 μmol·L^-1 exhibited no cytotoxicity and alleviated HG-triggered cell injury in a dose-dependent manner. Specifically, 20 μmol·L^-1 Sch B significantly suppressed cell apoptosis, decreased LDH leakage, reduced ROS accumulation and restored disturbed oxidative stress balance (P<0.01). Conclusion Sch B can ameliorate the damage of HG to PC12 cells, regulate oxidative stress, reduce cell apoptosis, and thus improve diabetic cognitive impairment.