The present study investigated the influence of sevoflurane on the cytotoxicity of neural stem cells of rats and deoxyribonucleic acid (DNA) methylation, and analyzed the correlation between degree of methylation and neurotoxicity of sevoflurane. Ten healthy Sprague-Dawley rats aged 6-8 weeks were randomly selected. The neural stem cells in the hippocampus of rats were isolated, followed by multiplication culture and induced differentiation. The nerve-related factors were observed and detected under a microscope. Moreover, the neural stem cells were treated with sevoflurane in different concentrations. Three wells were only added with the normal medium as the control group (C0), 3 wells were added with the low-concentration sevoflurane (0.2 g/ml) prepared by the medium as the low-concentration group (C1), 3 wells were added with the moderate-concentration of sevoflurane (0.5 g/ml) as the moderate-concentration group (C2), and 3 wells were added with the high-concentration sevoflurane (1 g/ml) as the high-concentration group (C3). The apoptosis rate was detected and calculated via Cell Counting Kit-8 (CCK-8) assay, the content of genomic DNA methylation in neural stem cells in each group was detected via high-performance liquid chromatography (HPLC), and the distribution of methylation in the chromosome in each group was compared. During the culture, neurospheres were produced, and the expression levels of four neural markers were increased. With the increase of sevoflurane concentration and the prolongation of time, the apoptosis rate of stem cells was increased. The content of methylation in cells treated with sevoflurane in a higher concentration was higher than that in other groups (P<0.05). According to the Pearsons correlation analysis, the content of methylation in neural stem cells was directly proportional to the concentration of sevoflurane. Methylation mostly occurred in the autosome, and the content of methylation in the high-concentration group was higher than those in the moderate-concentration, low-concentration and control groups (P<0.05). In conclusion, the concentration of sevoflurane can affect the degree of methylation in neural stem cells of rats and produce certain cytotoxicity.