The inbred genetic hypercalciuric stone-forming (GHS) rats exhibit many features of human idiopathic hypercalciuria and have elevated levels of vitamin D receptors (VDR) in calcium (Ca) transporting organs. On a normal Ca diet, 1,25(OH)₂D₃ (1,25D) increases urine (U) Ca to a greater extent in GHS than in controls (SD). The additional UCa may result from an increase in intestinal Ca absorption and/or bone resorption. To determine the source, we asked whether 1,25D would increase UCa in GHS fed a low Ca (0.02%) diet. With 1,25D, UCa in SD increased from 1.2±0.1 to 9.3±0.9 mg/d and increased more in GHS from 4.7±0.3 to 21.5±0.9 mg/d (p<0.001). In GHS rats on LCD with or without 1,25D, UCa far exceeded daily Ca intake (2.6 mg/d). While the greater excess in UCa in GHS rats must be derived from bone mineral, there may also be a 1,25D-mediated decrease in renal tubular Ca reabsorption. RNA expression of the components of renal Ca transport indicated that 1,25D administration results in a suppression of klotho, an activator of the renal Ca reabsorption channel TRPV5, in both SD and GHS rats. This fall in klotho would decrease tubular reabsorption of the 1,25D-induced bone Ca release. Thus the greater increase in UCa with 1,25D in GHS fed LCD strongly suggests that the additional UCa results from an increase in bone resorption, likely due to the increased number of vitamin D receptors in the GHS rat bone cells, with a possible component of decreased renal tubular calcium reabsorption.