Furosemide is a loop diuretic agent which has been used to treat hypercalcemia because it increases renal calcium excretion. The effect of furosemide on calcium transport molecules in distal tubule has yet to be investigated. We conducted studies to examine the effect of furosemide on renal calcium excretion and expression of calcium transport molecules in mice. Mice were administered with a single dose of furosemide (15mg/kg) and examined 4 hours later, or with twice daily furosemide injections for 3 days. To evaluate the effect of volume depletion, salt was supplemented in the drinking water. Our results showed that in acute experiments, furosemide enhanced urinary calcium excretion which was associated with a significant increase in mRNA levels of TRPV5, TRPV6 and calbindindin-D28k, but not calbindin-D9k as measured by real-time RT-PCR. Chronic furosemide administration induced 3-4-fold increase in urinary calcium excretion as well as elevated mRNA levels of TRPV5, TRPV6, calbindin-D28k and calbindin-D9k without or with salt supplement. Similar upregulation of calcium transport molecules was observed in mice with gentamicin-induced hypercalciuria. Co-administration of chlorothiazide decreased furosemide-induced calciuria, either acutely or chronically, but still accompanied with upregulation of these transport molecules. Immunofluorescent staining studies revealed comparable increased protein abundance in TRPV5 and calbindin-D28k. We conclude that furosemide treatment enhances urinary calcium excretion. Increased abundance of calcium transport molecules in the DCT represents a solute load dependent effect in response to increased calcium delivery and serves as a compensatory adaptation in the downstream segment.