Key points Adiponectin is a regulator of skeletal muscle mitochondrial biogenesis. Previous research using the ob/ob mouse (leptin deficient, low adiponectin) has suggested that the presence of adipokines, including adiponectin, is necessary for exercise‐induced increases in mitochondrial content. In the current study, we examined the importance of adiponectin as a regulator of skeletal muscle mitochondrial content in response to exercise by comparing wildtype and adiponectin deficient mice. Adiponectin deficient mice showed no differences in resting VO2, RER, or time to exhaustion during exercise when compared to wildtype mice. There were no differences in various protein markers of mitochondrial content. A single bout of treadmill running increased the mRNA expression of mitochondrial proteins similarly in wildtype and adiponectin deficient mice. Chronic exercise (8 weeks) also increased the protein content of mitochondrial markers similarly in wildtype and adiponectin deficient mice. We conclude that Ad is not required for exercise‐induced increases in muscle mitochondrial proteins. Abstract Adiponectin (Ad) has been proposed to be a regulator of mitochondrial biogenesis in skeletal muscle, and necessary for exercise‐induced increases in mitochondrial content. We first confirmed that Ad could acutely increase the expression of mitochondrial proteins during a 10 h incubation in isolated soleus and extensor digitorum longus (EDL) muscles. Next, we further examined the role of Ad as a regulator of mitochondrial content using Ad knockout (AdKO) mice. The AdKO animals showed no differences in resting VO2, respiratory exchange ratio, or in time to exhaustion during exercise when compared to wild‐type (WT) mice. There was a reduction in resting palmitate oxidation in isolated soleus from AdKO animals (−23%, P < 0.05) but not EDL, and 5‐aminoimidazole‐4‐carboxamide (AICAR)‐stimulated palmitate oxidation was similar in both genotypes regardless of muscle. There were no differences in protein markers of mitochondrial content (COX4, CORE1, CS, PDHE1α) in red and white gastrocnemius between WT and AdKO animals. A single bout of treadmill running increased the phosphorylation of AMP‐activated protein kinase (AMPK) and the mRNA expression of mitochondrial proteins in red and white gastrocnemius in both WT and AdKO animals, with no differences between genotypes. Finally, 8 weeks of chronic exercise training increased the protein content of mitochondrial markers similarly (∼25–35%) in red gastrocnemius from both WT and AdKO mice. Collectively, our results demonstrate that the absence of Ad is not accompanied by reductions in mitochondrial protein content, or a reduction in aerobic exercise capacity. We conclude that Ad is not required for the maintenance of mitochondrial content, or for exercise‐induced increases in skeletal muscle mitochondrial proteins.