Abstract Introduction Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with a gender bias towards major prevalence in male individuals. Several data suggest the involvement of oxidative stress and mitochondrial dysfunction in its pathogenesis, though differences between genders have not been evaluated. For this reason, we analysed features of mitochondrial oxidative metabolism, as well as mitochondrial chain complex enzyme activities and protein expression, lipid profile, and protein oxidative stress markers, in the Cu,Zn superoxide dismutase with the G93A mutation (hSOD1-G93A)- transgenic mice and Neuro2A(N2A) cells overexpressing hSOD1-G93A. Results and Conclusions Our results show that overexpression of hSOD1-G93A in transgenic mice decreased efficiency of mitochondrial oxidative phosphorylation, located at complex I, revealing a temporal delay in females with respect to males associated with a parallel increase in selected markers of protein oxidative damage. Further, females exhibit a fatty acid profile with higher levels of docosahexaenoic acid at 30 days. Mechanistic studies showed that hSOD1-G93A overexpression in N2A cells reduced complex I function, a defect prevented by 17β-estradiol pretreatment. In conclusion, ALS-associated SOD1 mutation leads to delayed mitochondrial dysfunction in female mice in comparison with males, in part attributable to the higher oestrogen levels of the former. This study is important in the effort to further understanding of whether different degrees of spinal cord mitochondrial dysfunction could be disease modifiers in ALS.