ATPase inhibitory factor 1 is a myokine inhibiting the hydrolytic activity of mitochondrial adenosine triphosphate synthase and ecto-F1-ATPase on the surface of many cells. IF1 affects ATP metabolism in mitochondria and the extracellular space and upregulates glucose uptake in myocytes; these processes are essential in physical activity. It is unknown whether the IF1 serum concentration is associated with exercise capacity. This study explored the association between resting IF1 serum concentration and exercise capacity indices in healthy people. IF1 serum concentration was measured in samples collected at rest in 97 healthy amateur cyclists. Exercise capacity was assessed on a bike ergometer at the successive stages of the progressive cardiopulmonary exercise test (CPET). IF1 serum concentration was negatively and significantly correlated with oxygen consumption, oxygen pulse, and load at various CPET stages. A better exercise capacity was associated with lower circulating IF1. IF1 may reflect better cellular/mitochondrial energetic fitness, but there is uncertainty regarding how IF1 is released into the intravascular space. We speculate that lower IF1 concentration may reflect a better cellular/mitochondrial integrity, as this protein is bound more strongly with ATPases in mitochondria and cellular surfaces in people with higher exercise capacity.