Human clear cell renal cell carcinoma (CCC) remains resistant to treatments despite the progress in targeted therapies. Several signaling pathways acting during renal development are reactivated during kidney tumorigenesis; this is the case of the sonic hedgehog (SHH)-Gli. Interestingly, the precursor of active vitamin D3 (VD3), cholecalciferol, has been demonstrated to be a strong inhibitor of SHH-Gli signaling. Here, we show the preclinical efficacy of cholecalciferol in CCC both in vitro and in vivo. A panel of CCC cell lines, tumors and normal corresponding tissues from CCC patients were used to evaluate the expression of the VD3 receptor and metabolizing enzymes and the effects of cholecalciferol treatment. Subsequently, xenografted mice were treated with cholecalciferol in a prophylactic or therapeutic manner; their response and the adverse effects were evaluated on the basis of weekly monitoring, followed by blood collection procedures and X-ray micro-computed tomography. VD3 receptor and metabolizing enzymes are dramatically decreased in human cell lines and tumors. Cholecalciferol decreases cell proliferation and increases cell death by inhibition of the SHH-Gli pathway. Xenografted mice treated with cholecalciferol exhibit absence of tumor development or substantial growth inhibition. The treatment was shown to be safe; it did not induce calcification or calcium reabsorption. These findings establish that, although VD3 receptors and metabolizing enzymes are absent in CCC, cholecalciferol supplementation is a strong tool to block the reactivation of SHH-Gli pathway in this pathology, leading ultimately to tumor regression. Cholecalciferol may have highly therapeutic potential in CCC.