Titanium alloy (Ti6Al4V) has widespread medical applications because of its excellent biocompatibility. Its biological responses can further be enhanced by polishing and passivation. Unfortunately, preparing titanium alloy samples of nanometric roughness is by far much more difficult than preparing those of micrometric roughness, and numerous investigations on roughness induced effects are all focused on micrometric scales. For the remedy, we investigate, at nanometric scale, the influence of roughness on surface properties and biological responses. Six groups of Ti6Al4V with average roughness (R(a)) values of 2.75-30.34 nm are prepared. The results indicated that nanometric roughness of samples change the wettability and amphoteric OH groups. The contact angles monotonically decrease from 2.75 to 30.34 nm and the rougher surfaces lead to higher wettability. The in vitro cell-culture studies, using Murine NIH-3T3 fibroblasts, showed the spindle-shaped morphology on rougher surface compared to round∕spherical morphology on smoother surface. A cytodetacher is employed to quantitatively measure the initial adhesion force of fibroblasts to specimen. The adhesion strength of fibroblasts, ranging from 55 to 193 nN, is significantly influenced by the nanometric roughness while the surface is within the range of 2.75-30.34 nm R(a) roughness, and the adhesion strength appeared stronger for rougher surface. The cell number on the smoother surface is higher than on the rougher surface at 5-day culture. The studies indicated that nanometric roughness would alter the surface properties and further influence cell morphology, adhesion strength, and proliferation.