The effects of microstructure and axial tension on the fatigue behavior of TC4 titanium alloy in high cycle (HCF) and very high cycle (VHCF) regimes are discussed in this paper. Ultrasonic three-point bending fatigue tests at 20 kHz were done on a fatigue life range among 10 5 -10 9 cycles of the alloys with equiaxed, bimodal and Widmanstatten microstructures. Experimental results without axial tension show that three typical shapes of S-N curves clearly present themselves for the three different microstructures. Moreover, the crack initiation sites abruptly shifted from surface to subsurface of the specimen in the very high cycle fatigue regime for equiaxed and bimodal microstructures. But for the Widmanstatten microstructure, both surface and subsurface crack initiation appeared in the high cycle fatigue regime, and the multi-points crack initiation was found in the bimodal microstructure. The subsurface fatigue crack originated from the α p grains in equiaxed and bimodal microstructures. However, it originated from the coarse grain boundary α in the Widmanstatten microstructure. Additionally, the S-N curve shape, fatigue life and fatigue crack initiation mechanism with axial tension are similar to that without axial tension. However, the crack origin point shifts inward with axial tension.