Testosterone undecanoate (T) treatment is common in female-to-male transsexuals (FtMs) but can induce impairment of mitochondrial function and oxidative stress. The effect of T treatment on the mitochondrial function and redox state of leukocytes of FtMs subjects was evaluated. This was an observational study conducted in a university hospital. Fifty-seven FtMs were treated with T (1,000 mg) for 12 weeks, after which anthropometric and metabolic parameters and mitochondrial function were evaluated. Anthropometric and metabolic parameters were evaluated. Mitochondrial function was studied by assessing mitochondrial oxygen (O2) consumption, membrane potential, reactive oxygen species (ROS) production, glutathione levels (GSH), and the reduced glutathione/oxidized glutathione (GSH)/(GSSG) ratio in polymorphonuclear cells. T treatment led to mitochondrial impairment in FtMs as a result of a decrease in mitochondria O2 consumption, the membrane potential, GSH levels, and the (GSH)/(GSSG) ratio and an increase in ROS production. Mitochondrial O2 consumption and membrane potential negatively correlated with T levels, which was further confirmed that the T treatment had induced mitochondrial dysfunction. T also produced a significant increase in total testosterone, free androgenic index, and atherogenic index of plasma, and a decrease in sex hormone-binding globulin and high-density lipoprotein cholesterol. Treatment of FtMs with T can induce impairment of mitochondrial function and a state of oxidative stress. This effect should be taken into account in order to modulate possible comorbidities in these patients.