Previous studies have suggested that Klotho provides reno-protection against unilateral ureteral obstruction (UUO)-induced renal tubulointerstitial fibrosis (RTF). Because the existing studies are mainly performed using heterozygous Klotho mutant (HT) mice, we focused on the effect of UUO on homozygous Klotho mutant (kl/kl) mice. UUO kidneys from HT mice showed a significantly higher level of RTF and TGF-β/Smad3 signaling than wild-type (WT) mice, whereas both were greatly suppressed in kl/kl mice. Primary proximal tubular epithelial culture cells isolated from kl/kl mice showed no suppression in TGF-β1-induced epithelial mesenchymal transition (EMT) compared to those from HT mice. In the renal epithelial cell line NRK52E, a large amount of inorganic phosphate (Pi), FGF23, or calcitriol was added to the medium to mimic the in vivo homeostasis of kl/kl mice. Neither Pi nor FGF23 antagonized TGF-β1-induced EMT. In contrast, calcitriol ameliorated TGF-β1-induced EMT in a dose dependent manner. A vitamin D3-deficient diet normalized the serum 1,25 (OH)2 vitamin D3 level in kl/kl mice and enhanced UUO-induced RTF and TGF-β/Smad3 signaling. In conclusion, the alleviation of UUO-induced RTF in kl/kl mice was due to the TGF-β1 signaling suppression caused by an elevated serum 1, 25(OH)2 vitamin D3.