Objective: Klotho is an aging-modulating protein expressed mainly in the kidneys and choroid plexus, which can also be shed, released into the circulation and act as a hormone. Klotho deficient mice are smaller compared to their wild-type counterparts and their somatotropes show marked atrophy and reduced number of secretory granules. Recent data also indicated an association between klotho levels and growth hormone (GH) levels in acromegaly. We aimed to study the association between klotho levels and GH deficiency (GHD) in children with growth impairment. Design: Prospective study comprising 99 children and adolescents (aged 9.0±3.7 years, 49 male) undergoing GH stimulation tests for short stature (height-SDS = -2.1±0.6). Klotho serum levels were measured using an α-klotho ELISA kit. Results: Klotho levels were significantly lower (p<0.001) among children with organic GHD (n = 11, 727±273 pg/ml) compared to both GH sufficient participants (n = 59, 1497±754 pg/ml) and those with idiopathic GHD (n = 29, 1645±778 pg/ml). The difference between GHS children and children with idiopathic GHD was not significant. Klotho levels positively correlated with IGF-1- standard deviation scores (SDS) (R = 0.45, p<0.001), but were not associated with gender, pubertal status, age or anthropometric measurements. Conclusions: We have shown, for the first time, an association between low serum klotho levels and organic GHD. If validated by additional studies, serum klotho may serve as novel biomarker of organic GHD.