Objective To evaluate the effect of inhibiting ras‑related C3 botulinum toxin substrate 1 (Rac1) expression on high glucose‑induced neurotoxicity. Methods PC12 cells at the logarithmic growth phase were seeded in 6‑well plates. According to the random number table method, they were divided into four groups (n=3): a normal glucose group (group C), a high glucose group (group HG), a high glucose+lentivirus inhibiting Rac1 small hairpin RNA group (group HG+shRac1) and a high glucose+lentivirus negative control group (group HG+NC). PC12 cells in group C were cultured in DMEM with 4.5 g/L glucose for 24 h, the cells in group HG were cultured in DMEM with 45 g/L glucose for 24 h, the cells in group HG+shRac1 were cultured in DMEM medium with 45 g/L glucose and transfected with lentiviral vector expressing shRNA of Rac1 for 24 h, and the cells in group HG+NC were transfected with negative lentiviral vector in DMEM medium containing a glucose concentration of 45 g/L for 24 h. Then, the levels of microtubule‑associated protein 1 light chain 3 (LC3)Ⅰ, LC3Ⅱ, Bcl‑2/adenovirus E1B protein‑interacting protein 3 (BNIP3) and p62 were detected by Western blot and the ratio of LC3Ⅱ/LC3Ⅰ was calculated. The cellular viability was detected by CCK8 assay. The apoptotic rate was measured by flow cytometry. The levels of cellular reactive oxygen species (ROS) was detected by dihydroethidium. Moreover, the relative expression of autophagy‑related gene 7 (Atg7), adenosinetriphosphatase 6 (ATPase6) and 60S ribosomal protein L13 (Rpl13) were detected by real‑time fluorescent polymerase chain reaction (PCR), and the ratio of ATPase6/Rpl13 was calculated. Results Compared with group C, group HG and group HG+NC showed increases in ROS production and apoptotic rate (P<0.05), and decreases in cellular viability (P<0.05). Compared with group HG+NC, group HG+shRac1 presented decreases in ROS production and apoptotic rate (P<0.05), and increases in cellular viability (P<0.05), up‑regulated Atg7 relative expression and BNIP3 protein levels (P<0.05), down‑regulated p62 protein levels (P<0.05), increased LC3Ⅱ/LC3Ⅰ ratio (P<0.05) and decreased ATPase6/Rpl13 ratio (P<0.05). Conclusions Inhibition of Rac1 can attenuate high glucose‑induced neurotoxicity, which may be associated with enhanced mitophagy.