In kidney, the epithelial Ca²⁺ channel TRPV5 constitutes the apical entry pathway in the process of active Ca²⁺ reabsorption. The regulation of Ca²⁺ influx through TRPV5 is of crucial importance, since it determines the final amount of Ca²⁺ excreted in the urine. The present study identifies FKBP52 as an auxiliary protein of TRPV5, inhibiting the channel activity. FKBP52 shows specific interaction with TRPV5 and both proteins co-localize in renal epithelial cells. On the functional level, FKBP52 decreases Ca²⁺ influx through TRPV5 as demonstrated in radioactive ⁴⁵Ca²⁺ uptake measurements and electrophysiological studies of TRPV5-expressing human embryonic kidney 293 cells. On the other hand, gene silencing of FKBP52 or administration of the FKBP52 blocker, FK506, enhances Ca²⁺ influx through TRPV5. The inhibitory action of FKBP52 on TRPV5 activity is blunted by mutation of its peptidyl-propyl cis-trans isomerase domain, showing that the FKBP52 catalytic property is critical for channel activity. In conclusion, these results suggest that FKBP52 plays an important role in the regulation of TRPV5 and thus in the process of Ca²⁺ reabsorption.