Serotonin (5-HT)--a monoamine with a variety of physiological functions--has recently emerged as a major regulator of bone mass. 5-HT is synthesized in the brain and the gut, and gut-derived 5-HT contributes to circulating 5-HT levels and is a negative modulator of bone mass and quality. 5-HT's negative effects on the skeleton are considered to be mediated via its receptors and transporter in osteoblasts and osteoclasts; however, 5-HT can also incorporate covalently into proteins via a transglutaminase-mediated serotonylation reaction, which in turn can alter protein function. Plasma fibronectin (pFN)--a major component of the bone extracellular matrix that regulates bone matrix quality in vivo--is a major transglutaminase substrate in bone and in osteoblast cultures. We have recently demonstrated that pFN assembly into osteoblast culture matrix requires a Factor XIII-A (FXIII-A) transglutaminase-mediated crosslinking step that regulates both quantity and quality of type I collagen matrix in vitro. In this study, we show that 5-HT interferes with pFN assembly into the extracellular matrix in osteoblast cultures, which in turn has major consequences on matrix assembly and mineralization. 5-HT treatment of MC3T3-E1 osteoblast cultures dramatically decreased both pFN fibrillogenesis as analyzed by immunofluorescence microscopy and pFN levels in DOC-soluble and DOC-insoluble matrix fractions. This was accompanied by an increase in pFN levels in the culture media. Analysis of the media showed covalent incorporation of 5-HT into pFN. Minor co-localization of pFN with 5-HT was also seen in extracellular fibrils. 5-HT also showed co-localization with FXIII-A on the cell surface and inhibited its transamidation activity directly. 5-HT treatment of osteoblast cultures resulted in a discontinuous pFN matrix and impaired type I collagen deposition, decreased alkaline phosphatase and lysyl oxidase activity, and delayed mineralization of the cultures. Addition of excess exogenous pFN to cultures treated with 5-HT resulted in a significant rescue of pFN fibrillogenesis as well as type I collagen deposition and mineralization. In summary, our study presents a novel mechanism on how increased peripheral extracellular 5-HT levels might contribute to the weakening of bone by directly affecting the stabilization of extracellular matrix networks.