Brain mitochondrial dysfunction and reduced testosterone levels are common features of aging in men. Although evidence suggests that the two phenomena are interrelated, it is unclear whether testosterone supplementation ameliorates mitochondrial dysfunction in the aging male brain. Here, we show that testosterone supplementation significantly alleviates exploratory behavioral deficits and oxidative damage in the substantia nigra and hippocampus of aging male rats. These effects were consistent with improved mitochondrial function, reflected by testosterone-induced increases in mitochondrial membrane potential (MMP), antioxidant enzyme (GSH-PX, catalase, and Mn-SOD) expression/activity, and mitochondrial respiratory complex activities in both brain regions. Furthermore, elevated PGC-1α, NRF-1, and TFAM expression (suggestive of enhanced mitochondrial biogenesis), increased citrate synthase activity, mtDNA copy number, and ND1, COX1, and ATP6 expression (indicative of increased mitochondrial content), as well as increased PINK1/Parkin and decreased P62 expression (suggesting mitophagy activation), were detected in the substantial nigra and hippocampus of aged male rats after testosterone supplementation. These findings suggest that testosterone supplementation may be a viable approach to ameliorating brain mitochondrial dysfunction and thus prevent or treat cognitive-behavioral deficits and neurodegenerative conditions associated with aging.