Background Amyotrophic lateral sclerosis (ALS) is an age-dependent and incurable neurodegenerative disease. It is pathologically characterized by selective degeneration of motor neurons resulting in a catastrophic loss of motor function. Previous studies showed that excess copper (Cu) might have a role in ALS etiology. However, the underlying mechanism remains incompletely elucidated. Therefore, we aimed to investigate the neurotoxicity and underlying mechanism of Cu exposure in female ALS mice with SOD1G93A gene mutation. Methods After three months of treatment with low-dose Cu exposure (0.13 PPM copper chloride drinking water), the motor ability was assessed by various behavioral methods, including climbing-pole test, rotarod test, hanging endurance test, grip strength test and gait analysis. Muscle atrophy and fibrosis were evaluated by histopathology. The proliferation of astrocytes and microglia was assessed by immunohistochemical analysis. The number of motor neurons in the spinal cord was determined by Nissl staining and ChAT immunohistochemical staining. Western blotting and proteomics revealed the effect of Cu exposure on mitochondrial protein expression in mice. Results Behavioral tests showed that Cu exposure worsened motor function in SOD1G93A mice. Compared with SOD1G93A transgenic mice, there was no significant difference in the pole-climbing test after Cu exposure (P =0.6156). There were significant changes in rotarod test, hanging endurance test and grip strength test (P =0.0121; P = 0.0085; P =0.0105). At the same time, Cu exposure aggravated muscle fibrosis and the loss of spinal motor neurons (P <0.05) and was associated with neuroinflammation (P <0.05). The proteomic analysis further showed that differentially expressed proteins among wild-type mice (WT), ALS mice, and Cu-treated ALS mice were mainly involved in muscle contraction, ATP metabolic processes, immune responses, peroxisomes, mitochondrial electrons respiratory transport chain, GMP biological processes, and were co-related with disturbances in energy metabolism, which was reflected in mitochondrial dysfunction in ALS mice. In addition, Western Blot showed that Cu exposure decreased the expression of mitochondria-related proteins (P <0.05), which was consistent with the results of proteomic analysis. Conclusions In conclusion, our findings showed that Cu exposure aggravates motor dysfunction and pathological changes in ALS mice via mitochondrial dysfunction.