Exercise-induced activation of adenosine monophosphate-activated protein kinase (AMPK) and substrate phosphorylation modulate the metabolic capacity of mitochondria in skeletal muscle. However, the key effector(s) of AMPK and the regulatory mechanisms remain unclear. Here, we showed that AMPK phosphorylation of the folliculin interacting protein 1 (FNIP1) serine-220 (S220) controls mitochondrial function and muscle fuel utilization during exercise. Loss of FNIP1 in skeletal muscle resulted in increased mitochondrial content and augmented metabolic capacity, leading to enhanced exercise endurance in mice. Using skeletal muscle-specific nonphosphorylatable FNIP1 (S220A) and phosphomimic (S220D) transgenic mouse models as well as biochemical analysis in primary skeletal muscle cells, we demonstrated that exercise-induced FNIP1 (S220) phosphorylation by AMPK in muscle regulates mitochondrial electron transfer chain complex assembly, fuel utilization, and exercise performance without affecting mechanistic target of rapamycin complex 1-transcription factor EB signaling. Therefore, FNIP1 is a multifunctional AMPK effector for mitochondrial adaptation to exercise, implicating a mechanism for exercise tolerance in health and disease.