Experimental data have shown that VDR overexpression in the duodenum and kidney cortex is a biological characteristic of genetic hypercalciuric stone-forming rats (GHS rat), and a link between idiopathic calcium stone formation and the microstatellite marker D12S339 (near the VDR locus) has been proven in humans. Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin-D28k and PMCA1b in NRK cell lines. VDR knockdown results in a decrease in intracellular Ca2+ concentration in NRK cell lines. The effect of the elevated VDR level in the kidney on hypercalciuria and the underlying mechanisms need to be further addressed. OBJECTIVE center dot To determine the effects of vitamin D receptor (VDR) on hypercalciuria and the mechanisms underlying such effects. MATERIALS AND METHODS center dot The adenovirus vector-delivered microRNA targeting rat VDR was constructed. We infected the normal rat kidney epithelial cell line NRK (Cellbank, China) with the adenovirus and then collected the cells at 0, 48, 72, 96, 120 h after infection. The mRNA and protein levels of VDR and VDR-dependent epithelial Ca2+ transport proteins were detected using real-time polymerase chain reaction and Western blot assays, respectively. center dot Fluorescent Ca2+ indicator Fluo-4 NW (Fluo-4 NW calcium assay kit, Molecular Probes, Invitrogen, USA) and laser scanning confocal microscope (Olympus, FV500-IX71, Japan) were used to detect the cytosolic free Ca2+ concentration at different time points after infection. RESULTS center dot The mRNA and protein level of VDR, transient receptor potential vanilloid receptor subtype 5 (TRPV5), calbindin-D-28k and plasma membrane Ca2+-ATPase (PMCA1b) in infected NRK cells was significantly lower at 72 and 96 h after infection than that in control cells. center dot There was no significant difference between the two groups in the mRNA and protein level of TRPV6 and the Na+/Ca2+-exchanger (NCX1). center dot Furthermore, VDR knockdown results in a decrease in intracellular Ca2+ concentration ([Ca2+]i) in NRK cell lines. CONCLUSIONS center dot Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin-D28k and PMCA1b, but not of TRPV6 or NCX1, in NRK cell lines. VDR knockdown results in a decrease in [Ca2+]i in NRK cell lines. center dot The effect of the elevated VDR level in the kidney on hypercalciuria and the mechanisms underlying need to be further addressed.