Background: The CKD-associated decline in soluble α-Klotho (α-Klotho) levels is considered detrimental. Some studies suggest a direct induction of α-Klotho concentrations by growth hormone (GH). In the present study, the effect of exogenous GH administration on α-Klotho concentrations in a clinical cohort with mild chronic kidney disease (CKD) and healthy subjects was studied. Methods: A prospective, single-center open case-control pilot study was performed involving 8 patients with mild CKD and 8 healthy controls matched for age and sex. All participants received subcutaneous GH injections (Genotropin®, 20 mcg/kg/day) for 7 consecutive days. α-Klotho concentrations were measured at baseline, after 7 days of therapy and 1 week after the intervention was stopped. Results: α-Klotho concentrations were not different between CKD-patients and healthy controls at baseline (554 (388-659) vs. 547 (421-711) pg/mL, P = 0.38). Overall, GH therapy increased α-Klotho concentrations from 554 (405-659) to 645 (516-754) pg/mL, P < 0.05). This was accompanied by an increase of IGF-1 concentrations from 26.8 ± 5.0 nmol/L to 61.7 ± 17.7 nmol/L (P < 0.05). GH therapy induced a trend toward increased α-Klotho concentrations both in the CKD group (554 (388-659) to 591 (358-742) pg/mL (P = 0.19)) and the healthy controls (547 (421-711) pg/mL to 654 (538-754) pg/mL (P = 0.13)). The change in α-Klotho concentration was not different for both groups (P for interaction = 0.71). α-Klotho concentrations returned to baseline levels within one week after the treatment (P < 0.05). Conclusions: GH therapy increases α-Klotho concentrations in subjects with normal renal function or stage 3 CKD. A larger follow-up study is needed to determine whether the effect size is different between both groups or in patients with more severe CKD. Trial registration: This trial is registered in EudraCT (2013-003354-24).