Pentraxin 3 (PTX3) is a multifunctional glycoprotein regulating inflammatory response, cell proliferation and migration and deposition and remodelling of the extracellular matrix by a variety of cells. In this study, we investigated the possible role of PTX3 in bone homeostasis. To this end, we compared the expression and function of PTX3 in human osteoblasts of osteoporotic, osteoarthritic patients and young subjects not affected by bone diseases. Immunohistochemical analysis performed on bone head biopsies showed a close association between bone health and the number of osteoblasts expressing PTX3. Noteworthy, the proportion of PTX3-positive osteoblasts resulted to be significantly lower in osteoporotic patients compared with both young patients and osteoarthritic patients of the same age. Ex vivo culture of osteoblasts isolated from the three groups of patients confirmed in vivo observation. Specifically, we observed rare runt-related transcription factor 2 (RUNX2) immunopositive osteoblasts expressing PTX3 in cell cultures derived from osteoporotic patients and western blotting analysis showed 80% reduction of PTX3 in the corresponding culture extracts compared with young and osteoarthritic patients. The treatment of human osteoblast primary cultures derived from young patients with anti-PTX3 antibody dramatically affected osteoblast behaviour. Indeed, they lost the morphological and molecular features typical of mature osteoblasts, acquiring fibroblast-like shape and highly decreasing nuclear factor kappa-B ligand (RANKL) and RUNX2 expression. Also, the inhibition of PTX3 negatively affected osteoblast proliferation and their ability to form cell clusters and microhydroxyapatite crystals. Altogether, these results suggest a central role of PTX3 in bone homeostasis showing its involvement in osteoblast proliferation, differentiation and function.