1,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3] is a critical regulator of bone development. Protein disulfide isomerase A3 (Pdia3) is a multifunctional protein that has been associated with rapid membrane-initiated signalling by 1alpha,25(OH)2D3 in several cell types. To identify the physiological roles of Pdia3 in skeletal development, we generated Pdia3-deficient mice. No homozygous mice were observed at birth, indicating that the targeted disruption of the Pdia3 gene resulted in embryonic lethality. Pdia3 deficiency also resulted in skeletal manifestations as revealed by muCT analysis of femurs from 15-week-old heterozygous mice. The Pdia3+/- mice had increased metaphyseal bone volume and trabeculae compared to Pdia3+/+ mice. In contrast, the area and thickness of cortical bone at the femoral mid-diaphysis of Pdia3+/+ mice significantly exceeded that of Pdia3+/- mice. In vitro studies in osteoblast-like MC3T3-E1 cells showed that silencing of Pdia3 abolished 1alpha,25(OH)2D3-induced rapid activation of protein kinase C (PKC) while overexpression of Pdia3 resulted in augmentation of PKC activity by 1alpha,25(OH)2D3. Taken together, these data indicated that Pdia3 plays a crucial role in 1alpha,25(OH)2D3-regulated bone formation and the Pdia3-PKC signalling pathway might be involved in this process.