1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃], a key regulator of mineral metabolism, regulates expression of several genes related to bone formation. The present study examined the 1,25(OH)₂D₃-mediated regulation of natriuretic peptide receptor-C (NPR-C) expression in osteoblasts. 1,25(OH)₂D₃ treatment significantly increased NPR-C-dependent atrial natriuretic peptide-binding activity and synthesis of the NPR-C protein in mouse osteoblastic cells in a cell-specific manner. Western blot analysis also demonstrated that 1,25(OH)₂D₃ upregulated expression of NPR-C protein in slow kinetics. Next, Northern blot analysis revealed a significant increase in the steady-state NPR-C mRNA level by 1,25(OH)₂D₃. Sequence analysis of the 9 kb of the 5'-flanking region of the mouse NPR-C gene revealed an absence of consensus vitamin D-response elements, and promoter analysis using osteoblastic cells stably transfected with mouse NPR-C promoter-reporter constructs showed a slight increase of promoter activity with 1,25(OH)₂D₃ treatment. In addition, a nuclear run-on assay exhibited that the transcriptional rate of the NPR-C gene was unchanged by 1,25(OH)₂D₃, whereas that of the osteopontin gene was increased. Evaluation of NPR-C mRNA half-life demonstrated that 1,25(OH)₂D₃ significantly increased the NPR-C mRNA stability in osteoblastic cells. 1,25(OH)₂D₃ attenuated intracellular cGMP production in osteoblastic cells stimulated by C-type natriuretic peptide (CNP) without a significant change of the natriuretic peptide receptor-B mRNA level, suggesting enhancement of the clearance of exogenously added CNP via NPR-C. Furthermore, NPR-C and osteopontin mRNAs in mouse calvariae were significantly increased by administration of 1,25(OH)₂D₃, and immunohistological analysis demonstrated that NPR-C is actually and strongly expressed in mouse periosteal fibroblasts. These findings suggest that 1,25(OH)₂D₃ can play a critical role for determination of the natriuretic peptide availability in bones by regulation of NPR-C expression through stabilizing its mRNA.