A comprehensive investigation into dry sliding wear and associated evolutions of subsurface microstructure and nano-mechanical properties of a bulk nano-grained Ti6Al4V alloy (NG-Ti6Al4V) was conducted. Comparing with its coarse-grained counterpart (CG-Ti6Al4V), NG-Ti6Al4V always exhibited lower friction coefficient and greater wear resistance in a wide range of tribological conditions. It was also found that the sliding wear induced nano-mechanical properties and microstructure evolutions in Ti6Al4V alloy were largely dependent on its initial microstructural features. For the first time, TEM observations provided a direct experimental evidence that the originally saturated nanostructure in NG-Ti6Al4V could be further refined by sliding wear. This study proposed an effective strategy to improve the load-bearing ability of Ti6Al4V alloy by producing bulk homogeneous nanostructure.