We hypothesized that enhanced skeletal muscle mitochondrial function following aerobic exercise training is related to an increase in mitochondrial transcription factors, DNA abundance (mtDNA) and mitochondria-related gene transcript levels as well as spontaneous physical activity levels (SPA). We report the effects of daily treadmill training on 12 weeks old FVB mice for 5 days/week over 8 weeks at 80% peak VO₂ and studied the training effect on changes in body composition, glucose tolerance, muscle mtDNA muscle, mitochondrial-related gene transcripts, in vitro muscle mitochondrial ATP production capacity (MATPC) and SPA levels. Compared with the untrained mice, the trained mice had higher VO₂ peak (+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% to 43.8%: p< 0.04) and MATPC (+28.9% to +32.4%: p<0.01). Gene array analysis showed significant differences in mRNAs of mitochondrial 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 gamma coactivator 1 alpha (PGC-1) protein (+99.5% p <0.04) and mitochondrial transcription factor A (TGFA) 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, mitochondrial DNA abundance, mitochondria-related gene transcript levels and mitochondrial function, and that this enhancement in mitochondrial function occurs in association with increased spontaneous physical activity.