Abstract Podoplanin is the only counter-receptor of platelet CLEC-2 and is expressing on mature osteoblast, but there is no report on the role of podoplanin and CLEC-2 in calcification. This study aimed to investigate the role of podoplanin binding to CLEC-2 in the calcification of osteoblasts carrying homozygously deleted Pdpn alleles ( Pdpn Δ/Δ ) by heterozygously expressing collagen type I alpha 1 promoter ( Col1a )-driven Cre recombinase. There were no macroscopic abnormalities in the bone and dentin of Col1a11-Cre;Pdpn Δ/Δ mice but the coccygeal bone medullary cavity was very narrow. In the quantitative analysis for alizarin red-stained products and alkaline phosphatase activities on the cultured calvarial osteoblasts, the amounts of calcified products and alkaline phosphatase activity of calvarial osteoblasts of both Pdpn fl/fl and Col1a11-Cre;Pdpn Δ/Δ mice were significantly higher in the calcification medium than in the α-mem. Both the amounts of calcified products and alkaline phosphatase activity of calvarial osteoblasts from Pdpn fl/fl mice were significantly lower in the calcification medium with CLEC-2 than without CLEC-2 while there were no significant differences in the amounts of calcified products and alkaline phosphatase activities of calvarial osteoblasts from Col1a11-Cre;Pdpn Δ/Δ mice with CLEC-2. Platelet CLEC-2 may play a role in regulating the calcification via binding to podoplanin on mature osteoblasts expressing podoplanin in the medullary cavity of a part of the bone.