Abstract Background Klotho, a single-pass transmembrane protein primarily expressed in the kidneys, parathyroid glands, and choroid plexus of the brain, has a short cytoplasmic tail and a long extracellular domain, which can be cleaved and released as a soluble form. However, information regarding the origins and kinetics of soluble serum Klotho remains poorly understood. We evaluated serial changes in serum Klotho levels among living donors before and after retroperitoneoscopic nephrectomy as well as in their renal transplant recipients. Methods The levels of soluble Klotho in serum obtained from 10 living donors and their renal transplant recipients were determined using a sandwich enzyme-linked immunosorbent assay system. Results Serum soluble Klotho was detectable in all subjects. The baseline serum Klotho concentrations in the living donors ranged from 726.4 to 1417.1 pg/mL (median, 909.8 pg/mL; interquartile ranges IR, 754.8–1132.4), whereas that in the concomitant renal transplant recipients ranged from 397.5 to 1047.2 pg/mL (median, 613.0 pg/mL; IR, 445.9–750.8; P = .003). The levels of soluble serum Klotho measured 5 days after retroperitoneoscopic nephrectomy (median, 619.0 pg/mL; IR, 544.6–688.5; P = .001) were significantly lower than the baseline values. Among the renal transplant recipients, no significant changes in serum Klotho levels were observed during the observation period. Conclusion Our data regarding soluble serum Klotho levels obtained from living donors support the idea that the kidneys are a major source of soluble serum Klotho in human subjects without a deterioration of renal function. In recipients, concomitant acute kidney injuries and immunosuppressive protocols might modulate the release of soluble Klotho from the grafts into the circulation.