Microstructure evolution and wear behavior of Ti6Al4V alloy under cryogenic dry sliding wear condition was investigated. An external cryogenic device was designed to produce a steady cryogenic environment (−172 °C) during the wear process. We found that the near surface microstructure of Ti6Al4V alloy exhibited obvious accumulation of dislocations in the grain boundaries of α phase, as well as the refinement of α and β phase grains during cryogenic temperature wear. On the contrary, the plastic deformation was predominated by twins during room temperature wear. Under cryogenic temperature wear, little plastic deformation of microstructure was observed in the near surface and subsurface of specimens, while severe deformation was found to be prevalent under room temperature wear. Worn surfaces of specimens after cryogenic wear tests were smoother with lower block spalling and friction coefficient. The cryogenic wear behavior was considered to be fracture spalling of surface induced by the initiation and extension of fatigue cracks, unlike the severe oxidative and delamination wear under room temperature. Meanwhile, as compared to room temperature wear, wear rate of Ti6Al4V alloy was reduced by 49.7%, 57.2% and 36.6% under the loading force of 100 N, 150 N and 200 N respectively after cryogenic temperature wear.