Designing the bone implant surface by mimicking the structure of natural tissue is an intriguing means to achieve better osseointegration. In this study, a biomimetic surface with sparsely distributed 80 nm diameter nanotubes (SNT) with a spacing of about 20–80 nm from each other, contrary to the closely distributed nanotubes on pure titanium, is fabricated by anodization of near β titanium alloy Ti-5Zr-3Sn-5Mo-15Nb (TLM). The structure is more similar to the cross-section of collagen fibrils than commonly reported nanotubes on pure titanium. The SNT-textured Ti-alloy also shows good wettability and low elastic modulus more compatible with bone tissues. The surface bioactivity is evaluated in vitro by primary osteoblast cultures. Compared with the polished non-textured TLM, the SNT texture exhibits satisfactory bioactivity with protein adsorption, initial cell adhesion, cell differentiation as revealed by ALP activity and osteogenesis-related gene expression. Although cell proliferation is slightly suppressed, ECM deposition is enhanced. The enhanced cell functions are probably related to the biomimetic structure, biochemical characteristics, and mechanical properties of SNT-textured Ti-alloy as well as the potential fluid exchange effect among the nanotubes. The SNT texture which constitutes a bio-interface with fluid supply from the implant surface can be tailored to enhance biological properties such as cell immigration and differentiation.