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Titanium and titanium alloys are widely used as a biomaterial due to their mechanical strength, corrosion resistance, low elastic modulus, and excellent biocompatibility. TiO 2 nanotubes have excellent bioactivity, stimulating the adhesion, proliferation of fibroblasts and adipose-derived stem cells, production of alkaline phosphatase by osteoblasts, platelets activation, growth of neural cells and adhesion, spreading, growth, and differentiation of rat bone marrow mesenchymal stem cells. In this study, we investigated the functionality of fibroblast on titania nanotube layers annealed at different temperatures. The titania nanotube layer was fabricated by potentiostatic anodization of titanium, then annealed at 300, 530, and 630 °C for 5 h. The resulting nanotube layer was characterized using SEM (Scanning Electron Microscopy), TF-XRD (Thin-film X-ray diffraction), and contact angle goniometry. Fibroblasts viability was determined by the CellTiter-Blue method and cytotoxicity by Lactate Dehydrogenase test, and the cell morphology was analyzed by scanning electron microscopy. Also, cell adherence, proliferation, and morphology were analyzed by fluorescence microscopy. The results indicate that the modification in nanotube crystallinity may provide a favorable surface fibroblast growth, especially on substrates annealed at 530 and 630 °C, indicating that these properties provide a favorable template for biomedical implants.