Gradient structure (GS) is commonly designed and processed in engineering materials to improve mechanical properties especially fatigue performance by taking advantage of the strengthened surface. However, whether the very-high-cycle fatigue (VHCF) property can be improved by GS is questioning due to the different crack initiation mechanisms between low-, high-cycle and VHCF. In this paper, GS of a Ti-6Al-4V alloy is generated by pre-torsion and characterized by electron backscatter diffraction. Then the VHCF behavior of the GS specimen is studied. The fractography and synchrotron radiation X-ray microtomography presented detailed characteristics of the internal crack initiation region in VHCF of the titanium alloy with GS. The results indicated that, in contrast to the low- and high-cycle regimes, the VHCF strength is reduced for the specimens with GS. Thus, the GS induced by pre-torsion cannot enhance the VHCF strength of the titanium alloy. This implies that VHCF test (property) is an important consideration for the microstructural designed materials. The graphical abstract is available in Supplementary information.