Two important criteria of an ideal biomaterial in the field of stem cells research are to regulate the cell proliferation without the loss of its pluripotency and to direct the differentiation into a specific cell lineage when desired. The present study describes the influence of TiO 2 nanofibrous surface structures on the regulation of proliferation and stemness preservation of adipose-derived stem cells (ADSCs). TiO 2 nanofiber arrays were produced in situ onto Ti-6Al-4V substrate via a thermal oxidation process and the successful fabrication of these nanostructures was confirmed by field emission scanning electron microscopy (FESEM), energy dispersive spectrometer (EDS), X-ray diffractometer (XRD), and contact angle measurement. ADSCs were seeded on two types of Ti-6Al-4V surfaces (TiO 2 nanofibers and flat control), and their morphology, proliferation, and stemness expression were analyzed using FESEM, AlamarBlue assay, flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) after 2 weeks of incubation, respectively. The results show that ADSCs exhibit better adhesion and significantly enhanced proliferation on the TiO 2 nanofibrous surfaces compared to the flat control surfaces. The greater proliferation ability of TiO 2 nanofibrous surfaces was further confirmed by the results of cell cycle assay. More importantly, TiO 2 nanofibrous surfaces significantly upregulate the expressions of stemness markers Sox-2, Nanog3, Rex-1, and Nestin. These results demonstrate that TiO 2 nanofibrous surfaces can be used to enhance cell adhesion and proliferation while simultaneously maintaining the stemness of ADSCs, thereby representing a promising approach for their potential application in the field of bone tissue engineering as well as regenerative therapies.