Owing to the role of insulin-like growth factor 1 (IGF-I) in cardiac hypertrophy and the ability of Klotho in inhibiting the IGF-I action, we investigated effects of exercise on plasma Klotho and IGF-I and their association with cardiac hypertrophy. In this study, 10 non-athlete and 10 athlete women underwent a Bruce test (acute exercise) and 12-weeks water aerobics training (chronic exercise). Electrocardiographic parameters, plasma IGF-I and Klotho levels were measured in different time courses. The exercise training could significantly increase left ventricular end-diastolic diameter index (LVEDDI) in the non-athletes. Plasma levels of IGF-I significantly increased following acute and chronic exercises. The Klotho levels at the baseline were higher in athletes than non-athletes and its levels significantly increased immediately after acute exercise in both groups. The Klotho levels significantly decreased in non-athletes 24 h after chronic exercise, but its level was still higher than the baseline in the athletes. We found positive and negative correlations between cardiac hypertrophy indexes (LVEDDI and left ventricular mass index) with respectively IGF-I and Klotho. In conclusion, we found a stimulatory effect of acute and chronic exercises on plasma IGF-I and Klotho and association of IGF-I with exercise-induced cardiac hypertrophy. Moreover, Klotho could act as a negative regulator for exercise-induced cardiac hypertrophy.