Purpose: To investigate whether human adipose-derived stem cells will form a functional bone-like matrix on titanium substrates. The behavior of human adipose-derived stem cells was examined when grown in either serum-free, xeno-free stem cell growth medium or osteogenic differentiation medium and cultured on either machined titanium (MTi) or on roughened alumina-blasted titanium (ABTi) discs. Materials and Methods: Cellular proliferation, extracellular mineralized matrix production, osteogenic-related protein production (RUNX2 and osteocalcin), and gene expression for pluripotency and self-renewal (TERT and OCT4) and osteogenic-related (MSX2, RUNX2, and BGLAP) genes were performed. Results: Human adipose-derived stem cells in serum-free medium (hADSC) proliferated at a higher rate compared with osteogenically differentiated cells (hOS-ADSC); however, the osteogenically committed cells produced more mineralized matrix on the titanium surfaces compared with either tissue culture plastic or the undifferentiated cells. The immunofluorescence analysis showed that human adipose-derived stem cells cultured in serum-free medium and osteogenic differentiation medium produced RUNX2 on both the machined titanium surface and on the alumina-blasted titanium surface after 7 days in culture. Only osteogenically differentiated cells produced osteocalcin after 21 days. Relative gene expression showed stable expression of MSX2, RUNX2, and BGLAP over time on all surfaces. Only osteogenically differentiated cells displayed osteogenic characteristics over time. Conclusion: This study confirmed that human adipose-derived stem cells could be successfully grown in serum-free, xeno-free culture medium suitable for clinical use. Adipose-derived stem cells thus show potential utility for bone regeneration in association with titanium surfaces. Keywords: biomaterials, bone-implant interactions, bone substitutes