We hypothesized that enhanced skeletal muscle mitochondrial function following aerobic exercise training is related to an increase in mitochondrial transcription factors, DNA abundance [mitochondrial DNA (mtDNA)], and mitochondria-related gene transcript levels, as well as spontaneous physical activity (SPA) levels. We report the effects of daily treadmill training on 12-wk-old FVB mice for 5 days/wk over 8 wk at 80% peak O2 consumption and studied the training effect on changes in body composition, glucose tolerance, muscle mtDNA muscle, mitochondria-related gene transcripts, in vitro muscle mitochondrial ATP production capacity (MATPC), and SPA levels. Compared with the untrained mice, the trained mice had higher peak O2 consumption (+18%; P < 0.001), lower percentage of abdominal (−25.4%; P < 0.02) and body fat (−19.5%; P < 0.01), improved glucose tolerance ( P < 0.04), and higher muscle mitochondrial enzyme activity (+19.5–43.8%; P < 0.04) and MATPC (+28.9 to +32.4%; P < 0.01). Gene array analysis showed significant differences in mRNAs of mitochondria-related ontology groups between the trained and untrained mice. Training also increased muscle mtDNA (+88.4 to +110%; P < 0.05), peroxisome proliferative-activated receptor-γ coactivator-1α protein (+99.5%; P < 0.04), and mitochondrial transcription factor A mRNA levels (+21.7%; P < 0.004) levels. SPA levels were higher in trained mice ( P = 0.056, two-sided t-test) and significantly correlated with two separate substrate-based measurements of MATPC ( P < 0.02). In conclusion, aerobic exercise training enhances muscle mitochondrial transcription factors, mtDNA abundance, mitochondria-related gene transcript levels, and mitochondrial function, and this enhancement in mitochondrial function occurs in association with increased SPA.