The alpha-Klotho gene was identified as a possible “aging-suppressor” agent that extends life span when overexpressed. However, little is known about the association of the body composition with the secreted protein form of the alpha-Klotho gene (S-Klotho). Therefore, the aim of this study was to analyze the association of body composition, including lean and fat mass as well as bone mineral density (BMD), with S-Klotho plasma levels in middle-aged sedentary adults. A total of 74 (39 women) middle-aged sedentary adults (53.7 +/- 5.1 years old; 75.7 +/- 14.0kg; 167.8 +/- 9.8cm) participated in the study. We measured weight and height, and we used dual-energy X-ray absorptiometry to measure fat mass and lean mass. We calculated the body mass index (BMI), fat mass index (FMI), and lean mass index (LMI). The S-Klotho plasma levels were measured in the ethylenediaminetetraacetic acid plasma using a solid-phase sandwich enzyme-linked immunosorbent assay. There was a strong positive association between LMI and S-Klotho plasma levels (beta=74.794, R-2=0.346, p<0.001), which persisted after controlling for age and gender as well as after additionally controlling for FMI. Significantly positive associations of BMI and BMD were also found with S-Klotho plasma levels (beta=33.981, R-2=0.125, p=0.002 and beta=858.194, R-2=0.058, p=0.041, respectively), which disappeared after controlling for LMI (beta=0.183, R-2=0.611, p=0.984 and beta=-379.426, R-2=0.617, p=0.290, respectively). FMI was not significantly associated with S-Klotho plasma levels. Our study shows that LMI is strongly associated with S-Klotho plasma levels and explains the associations of BMI and BMD with S-Klotho plasma levels in middle-aged sedentary adults.