Mitochondrial dysfunction in various tissues has been associated with numerous conditions including aging. In testes, aging induces atrophy and a decline in male reproductive function but the involvement of mitochondria is not clear. The purpose of this study was to examine whether the mitochondrial profile differed with (1) aging, and (2) 10-weeks of treadmill exercise training, in the testes of young (6 month) and old (24 month) Fischer-344 (F344) animals. Old animals exhibited significant atrophy (30 % decline; P < 0.05) in testes compared to young animals. However, relative mitochondrial content was not reduced with age and this was consistent with the lack of change in the mitochondrial biogenesis regulator protein, peroxisome proliferator-activated receptor gamma coactivator 1-alpha and its downstream targets nuclear respiratory factor-1 and mitochondrial transcription factor A. No effect was observed in the pro- or anti-apoptotic proteins, Bax and Bcl-2, respectively, but age increased apoptosis inducing factor levels. Endurance training induced beneficial mitochondrial adaptations that were more prominent in old animals including greater increases in relative mtDNA content, biogenesis/remodeling (mitofusin 2), antioxidant capacity (mitochondrial superoxide dismutase) and lower levels of phosphorylated histone H2AX, an early marker of DNA damage (P < 0.05). Importantly, these exercise-induced changes were associated with an attenuation of testes atrophy in older sedentary animals (P < 0.05). Our results indicate that aging-induced atrophy in testes may not be associated with changes in relative mitochondrial content and key regulatory proteins and that exercise started in late-life elicits beneficial changes in mitochondria that may protect against age-induced testicular atrophy.