Ca2+ ions play a fundamental role in many cellular processes, and the extracellular concentration of Ca2+ is kept under strict control to allow the proper physiological functions to take place. The kidney, small intestine, and bone determine the Ca2+ flux to the extracellular Ca2+ pool in a concerted fashion. Transient receptor potential (TRP) cation channel subfamily V, members 5 and 6 (TRPV5 and TRPV6) have recently been postulated to be the molecular gatekeepers facilitating Ca2+ influx in these tissues and are members of the TRP family, which mediates diverse biological effects ranging from pain perception to male aggression. Genetic ablation of TRPV5 in the mouse allowed us to investigate the function of this novel Ca2+ channel in maintaining the Ca2+ balance. Here, we demonstrate that mice lacking TRPV5 display diminished active Ca2+ reabsorption despite enhanced vitamin D levels, causing severe hypercalciuria. In vivo micropuncture experiments demonstrated that Ca2+ reabsorption was malfunctioning within the early part of the distal convolution, exactly where TRPV5 is localized. In addition, compensatory hyperabsorption of dietary Ca2+ was measured in TRPV5 knockout mice. Furthermore, the knockout mice exhibited significant disturbances in bone structure, including reduced trabecular and cortical bone thickness. These data demonstrate the key function of TRPV5 in active Ca2+ reabsorption and its essential role in the Ca2+ homeostasis.