Objective To investigate damage of double-stranded DNA by methamphetamine (METH) in various types of nerve cells. Methods For in vitro experiments, primary cortical neurons and HT-22, BV2, HMC3 and U-87 MG cells were treated with METH. Cell morphology was observed and γ-H2AX expression was detected by Western blot. For in vivo experiments, METH was administered to mice by intraperitoneal injection, and behavioral analysis was performed by the open field test. Morphological changes of neurons in the prefrontal cortex and hippocampus of were observed by HE staining. Double-stranded DNA damage was observed by immunofluorescence, and γ-H2AX expression was detected by western blotting. Results In vitro, after treatment with METH, the morphology of primary cortical neurons, and HT-22, BV2, HMC3 and U-87 MG cells had obviously changed, the synapses of cells were shortened or disappeared, cell bodies had shrunk, and gaps were wider. γ-H2AX expression was significantly increased. In vivo, after administration of METH, compared with the saline group, the movement trajectory and distance were significantly increased in the METH group. HE staining showed that neurons in the prefrontal cortex and hippocampus were markedly edematous and eosinophilic, and some neurons had degenerated. Immunofluorescence showed that double-stranded DNA damage in neurons in the prefrontal cortex and hippocampus of the METH group was significantly increased, and the fluorescence intensity was significantly enhanced compared with the saline group. Western blot showed that γ-H2AX expression in the prefrontal cortex and hippocampus of the METH group was significantly increased compared with the saline group. Conclusions METH damaged doublestranded DNA in the nervous system. METH induced a significant increase in γ-H2AX expression in primary cortical neurons HT-22, BV2, HMC3 and U-87 MG cells, the prefrontal cortex, and hippocampus. This study provides a theoretical basis to elucidate the mechanism of METH-induced neurotoxicity.