Malignant insulinoma is a rare form of cancer with a poor prognosis because of metastatic dissemination and untreatable hypoglycemia. Effective chemotherapy of patients who are not cured by surgery is needed. Calcitriol has known anticancer properties on different neoplastic cell lines, but no data are available regarding its activity on tumorigenic pancreatic beta-cells. We analyzed the in vitro effects of calcitriol on the murine insulinoma cell line betaTC(3) and primary cultures of human isolated islets and benign insulinoma. The effect of in vivo calcitriol administration on insulinoma of recombinant insulin/Simian virus 40 oncogene-expressing transgenic mice was also investigated. In betaTC(3), calcitriol induced growth inhibition; apoptosis; down-regulation of insulin gene expression; and nongenomic activation of the MAPK pathway. MAPK kinase inhibitor (UO126) and staurosporine reduced calcitriol-mediated betaTC(3) death, and down-regulation of insulin gene transcription was prevented by staurosporine but not UO126. Calcitriol significantly decreased insulin release and mRNA levels of human islets and insulinoma cells. Finally, recombinant insulin/Simian virus 40 oncogene-expressing transgenic mice treated with calcitriol showed reduced insulinoma volumes because of increased apoptosis of adenomatous cells. Together, these findings provide the rationale for testing the efficacy of calcitriol in the treatment of patients with solid beta-cell tumors.