The microstructural features and the fatigue propensities of interior crack initiation region for very-high-cycle fatigue (VHCF) of a Ti-6Al-4V alloy were investigated in this paper. Fatigue tests under different stress ratios of R=-1, -0.5, -0.1, 0.1 and 0.5 were conducted by ultrasonic axial cycling. The observations by SEM showed that the crack initiation of VHCF presents a fish-eye (FiE) morphology containing a rough area (RA), and the FiE and RA are regarded as the characteristic regions for crack initiation of VHCF. Further examinations by TEM revealed that a layer of nanograins exists in the RA for the case of R=-1, while nanograins do not appear in the FiE outside RA for the case of R=-1, and in the RA for the case of R=0.5, which is explained by the Numerous Cyclic Pressing model. In addition, the estimations of the fatigue propensities for interior crack initiation stage of VHCF indicated that the fatigue life consumed by RA takes a dominant part of the total fatigue life and the related crack propagation rate is rather slow.