alpha-Klotho, a multifunctional protein, has been demonstrated to protect tissues from injury via anti-oxidation and anti-inflammatory effects. The expression of alpha-klotho is regulated by several physiological and pathological factors, including acute inflammatory stress, oxidative stress, hypertension, and chronic renal failure. Exhaustive exercise has been reported to result in tissue damage, which is induced by inflammation, oxidative stress, and energy metabolism disturbance. However, little is known about the effects of exhaustive exercise on the expression of alpha-klotho in various tissues. To determine the effects, the treadmill exhaustion test in mice was performed and the mice were sacrificed at different time points following exhaustive exercise. Our results confirmed that the full-length (130 kDa) and shorter-form (65 kDa) alpha-klotho were primarily expressed in the kidneys. Moreover, we found that, except for the kidneys and brain, other tissues primarily expressed the shorter-form alpha-klotho, including liver, which was in contrast to previous reports. Furthermore, the shorter-form alpha-klotho was decreased immediately following the acute exhaustive exercise and was then restored to the pre-exercise level or even higher levels in the next few days. Our results indicate that alpha-klotho may play a key role in the body exhaustion and recovery following exhaustive exercise.