Successful bone regeneration on titanium (Ti) surfaces is a key process in dental implant treatment. Bone marrow mesenchymal stem cells (BMSCs) are fundamental cellular components of this process, and their early recruitment, proliferation, and differentiation into bone-forming osteoblasts are crucial. A proteoglycan (PG)-rich layer has been reported to exist between Ti surfaces and bones; however, the molecules that could potentially affect the formation of this layer remain unknown. Family with sequence similarity 20 member B (FAM20B) is a newly identified kinase that regulates the synthesis of glycosaminoglycans, an important component of the PG-rich layer. Because FAM20B is also closely associated with bone development, in this study, we examined the function of FAM20B in osteogenic differentiation of BMSCs on Ti surfaces. For this, BMSC cell lines with knocked down FAM20B (shBMSCs) were cultured on Ti surfaces. The results showed that the depletion of FAM20B reduced the formation of a PG-rich layer between the Ti surfaces and cells. The shBMSCs exhibited downregulated expression of osteogenic marker genes (ALP and OCN) and decreased mineral deposition. Moreover, shBMSCs reduced the molecular levels of p-ERK1/2, which plays an important role in MSC osteogenesis. The nuclear translocation of RUNX2, an important transcription factor for osteogenic differentiation, on the Ti surfaces is inhibited by the depletion of FAM20B in BMSCs. Moreover, the depletion of FAM20B reduced the transcriptional activity of RUNX2, which is important in regulating the expression of osteogenic genes. STATEMENT OF SIGNIFICANCE: Bone healing and regeneration on implanted titanium surfaces is a cell-material interaction. Such an interaction is enabled by bone marrow mesenchymal stem cells (BMSCs), and their early recruitment, proliferation, and differentiation into bone-forming osteoblasts are essential for bone healing and osseointegration. In this study, we found that the family with sequence similarity 20-B influenced the formation of a proteoglycan rich layer between BMSCs and the titanium surface and regulated the differentiation of BMSCs into bone-forming osteoblasts. We believe that our study contributes significantly to the further exploration of bone healing and osseointegration mechanisms on implanted titanium surfaces.