Transient receptor potential vallinoid 5 (TRPV5) and TRPV6 are the most Ca2+-selective members of the TRP superfamily and are essential for active Ca2+ (re)absorption in epithelia. However, little is known about intracellular proteins that regulate the activity of these channels. This study identified BSPRY (B-box and SPRY-domain containing protein) as a novel factor involved in the control of TRPV5. The interaction between BSPRY and TRPV5 by GST pull-down and co-immunoprecipitation assays was demonstrated. BSPRY showed co-localization with TRPV5 in mouse kidney. Expression of BSPRY resulted in a significant reduction of the Ca2+ influx in Madin-Darby Canine Kidney cells that stably express TRPV5 without affecting channel cell-surface abundance. Finally, BSPRY expression in kidney was increased in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice, suggesting an inverse regulation by vitamin D3. Together, these results demonstrate the physiologic role of the novel protein BSPRY in the regulation of epithelial Ca2+ transport via negative modulation of TRPV5 activity.