The molecular mechanisms underlying skeletal muscle mitochondrial adaptations induced by aerobic exercise (AE) are not fully understood. We have previously shown that AE induces mitochondrial adaptations in cardiac muscle, mediated by sympathetic stimulation. Since direct sympathetic innervation of neuromuscular junctions influences skeletal muscle homeostasis, we tested the hypothesis that β-adrenergic receptor (β-AR)-mediated sympathetic activation induces mitochondrial adaptations to AE in skeletal muscle. Male FVB mice were subjected to a single bout of AE on a treadmill (80% Vmax, 60 min) under β-AR blockade with ICI 118,551 (ICI) or vehicle, and parameters of mitochondrial function and morphology/dynamics were evaluated. An acute bout of AE significantly increased maximal mitochondrial respiration in tibialis anterior (TA) isolated fiber bundles, which was prevented by β-AR blockade. This increased mitochondrial function after AE was accompanied by a change in mitochondrial morphology towards fusion, associated with increased Mfn1 protein expression and activity. β-AR blockade fully prevented the increase in Mfn1 activity and reduced mitochondrial elongation. To determine the mechanisms involved in mitochondrial modulation by β-AR activation in skeletal muscle during AE, we used C2C12 myotubes, treated with the non-selective β-AR agonist isoproterenol (ISO) in the presence of the specific β-AR antagonist ICI or during protein kinase A (PKA) and Gα protein blockade. Our in vitro data show that β-AR activation significantly increases mitochondrial respiration in myotubes, and this response was dependent on β-AR activation through a Gα-PKA signaling cascade. In conclusion, we provide evidence for AE-induced β-AR activation as a major mechanism leading to alterations in mitochondria function and morphology/dynamics. β-AR signaling is thus a key-signaling pathway that contributes to skeletal muscle plasticity in response to exercise.