Ti-6Al-4V parts are usually hypothesized as isotropic mechanical properties during micro-machining operation. However, the crystal anisotropic mechanical property is an influential factor in the machining of crystals with different sizes and orientations. The hypothesis causes distinct variation in predicting machining force, and consequently the surface integrity cannot be exactly predicted in the micro-machining. Actually, Ti-6Al-4V parts are pseudo isotropy composed of grains with different orientations, elastic modulus, and hardness. Simultaneously, the initiation mode of plastic slip system varies with the grain orientation when the crystal is subjected to an applied load. It is challengeable to evaluate the effect of crystal orientation on its macroscopic properties, either by traditional experimental methods or macroscopic finite element analysis. Nano-indentation provides one novel method to investigate the plastic deformation of selected grains with specific orientations to characterize their mechanical response properties. This research aims to investigate the plastic behavior of α phase Ti-6Al-4V with different orientation grains by means of nano-indentation experiment and crystal plastic finite element method. In the study, single crystals of α phase are classified into “hard grains” (θ < 30°), “soft grains” (θ > 60°) and “normal grains” (30° < θ < 60°) by defining the crystal inclination angle (θ). The elastic modulus and hardness of hard grains are 9% and 28% larger than those of soft grains, respectively. This difference in mechanical properties depends on the slip system that dominates the plastic deformation behavior. The plastic deformation of soft grains is dominated by prismatic slip (48%), while hard grains are accommodated to plastic deformation by basal slip (51%). The average total cumulative shear strain of the soft grains is double that of the hard grains at a maximum load of 10 mN. Meanwhile, the sensitivity analysis is performed to evaluate the effects of the three sets of < a > slip systems and the two sets of < c + a > slip systems on plastic deformation with different grain orientations. The research contributes to the understanding of the plastic deformation mechanism in micro-machining and the exactly predicting the surface integrity generated from micro-machining.