Rapamycin, an immunosuppressive drug used to prevent rejection after kidney transplantation, influences phosphate homeostasis, induces insulin resistance and has been shown to prolong lifespan in animal models. Because Klotho is an aging‐suppressor gene controlling phosphate metabolism and insulin sensitivity, we investigated the influence of rapamycin on Klotho expression. A total of 100 kidney transplant recipients, 50 chronically treated with rapamycin and 50 with calcineurin inhibitors, were enrolled; 20 healthy subjects were employed as control. In the rapamycin group, serum phosphate was lower than in the CNI group with an increase in phosphate excretion and a reduction in its reabsorption. In addition, rapamycin increased insulin resistance as shown by HOMA index. Rapamycin treatment of an immortalized proximal tubular cell line induced the expression of Klotho, the phosphorylation of AKT in Ser473, downstream target of mTORC2 and the expression of RICTOR, mTORC2 main component. AKT inhibition reduced the rapamycin‐induced expression of Klotho. In vivo rapamycin treatment induced higher degree of RICTOR and AKT Ser473 expression directly correlating with long‐term rapamycin exposure, FEPO4 and HOMA index. In conclusion, our data would suggest that rapamycin may influence phosphate homeostasis and insulin resistance modulating Klotho expression through mTORC2 activation. Rapamycin influences phosphate homeostasis and insulin resistance modulating klotho expression in epithelial tubular cells and this event is mediated by mTORC2 through AKT phosphorylation.