In order to clarify the relationship between the microstructure and the small fatigue crack initiation behavior in Ti-6Al-4V alloy, the small fatigue crack initiated in Ti-6Al-4V alloy was observed in detail by using an atomic force microscopy (AFM) and an electron back scattered diffraction (EBSD). The fatigue crack in Ti-6Al-4V alloy initiated from the basal slip in a-phase. In addition, in comparison with Schmid factor of α-phase from which the fatigue crack initiated and other α-phases around the crack initiation area, Schmid factor in a-phase from which the fatigue crack initiated is the highest compared with other α-phases.