Physicochemical properties, regulated by various surface modifications, influence the biological performance of materials. The interaction between surface charge and biomolecules is key to understanding the mechanism of surface-tissue integration. The objective of this study was to evaluate the biological response to a nanoscale titanium surface after ultraviolet (UVC, λ = 250 ± 20 nm) irradiation and to analyze the effects via a physicochemical mechanism. The surface characteristics were evaluated by field-emission scanning electron microscopy, X-ray photoelectron spectroscopy, surface profilometry, and contact angle assay. In addition, we applied the zeta-potential, a direct method to measure the electrostatic charge on UV-treated and UV-untreated titanium nanotube surfaces. The effect of the Ti surface after UV treatment on the biological process was determined by analyzing bovine serum albumin (BSA) adsorption and osteoblast-like MG-63 early adhesion, morphology, cytoskeletal arrangement, proliferation, and focal adhesion. Compared to an anodized titanium nanotube coating, UV irradiation altered the contact angles on the control surface from 51.5° to 6.2° without changing the surface topography or roughness. Furthermore, titanium nanotubes after UV treatment showed a significant reduction in the content of acidic hydroxyl groups and held less negative charge than the anodized coating. With regard to the biological response, along with an enhanced capability to adsorb BSA, osteoblasts exhibited higher colonization and viability on the UV-treated material. The results suggest that UV treatment enhances the biocompatibility by reducing the electrostatic repulsion between biomaterials and biomolecules.