Osteoclast inhibitory lectin (OCIL) is a type II C-type lectin and binds NK cell-associated receptor Nkrp1d and sulfated glycosaminoglycans. OCIL is expressed by several cell types found in bone and inhibits osteoclast differentiation. To determine whether OCIL may have wider effects on bone metabolism, we examined the effects of recombinant soluble OCIL on cultured osteoblasts and pre-osteoblastic KUSA O cells. Although OCIL did not affect osteoblast proliferation or apoptosis, or the formation of alkaline phosphatase positive colonies in cultured bone marrow, OCIL profoundly inhibited mineralization by primary osteoblasts and KUSA O cells in vitro. Analysis of ascorbate-treated KUSA O cells showed that addition of OCIL reduced bone sialoprotein (BSP), osterix and osteocalcin mRNA expression, as well as alkaline phosphatase activity while, in contrast, expression of markers associated with the earlier stages of osteoblast maturation or the transcription factors Runx2, ATF4 and c-fos were not affected by OCIL treatment. Indeed, osteocalcin expression was strongly inhibited within 3 days in a dose-dependent manner, although after subsequent removal of OCIL, osteocalcin mRNA levels recovered within 4 days. OCIL treatment also reduced osteocalcin expression in BMP-2 stimulated C2C12 cells. In support of a role for OCIL in mineralization, OCIL anti-sense oligonucleotide treatment of KUSA O cells increased mineralization and osteocalcin expression. In addition, insulin-, dexamethasone- and IBMX-stimulated KUSA O cells undergo adipocyte differentiation and OCIL treatment greatly suppressed this process. Consistent with this, OCIL also reduced adiponectin and resistin mRNA expression in these cells. Our data indicate that OCIL reduces osteoblastic function in vitro and this may be due to an inhibitory effect on osteoblast maturation. In addition, the reduction of adipocyte formation in KUSA O cells by OCIL indicates that OCIL may have wider effects on the mesenchymal lineage that may be important for both bone metabolism and other connective tissue functions.