An ultrasonic surface rolling process (USRP) is a novel mechanical surface treatment technique for enhancing the fatigue performance of metallic materials. In this work, USRP with different repeated processing numbers was employed for enhancing the fatigue performance of a Ti-6Al-4V alloy. The effect of USRP on their surface integrity (including microstructure, surface quality, microhardness, and residual stress) were investigated, which were characterized by means of scanning electron microscope, transmission electron microscope, confocal laser scanning microscope, microhardness tester, and X-ray diffraction residual stress tester. Especially, a refined microstructure (grain size: ~100–400 nm) was formed on the topmost surface of twelve-repeat USRP specimen. Subsequently, the fatigue behavior of the specimens was investigated via rotating-bending fatigue tests, and the results suggested that USRP could effectively enhance the fatigue performance of the Ti-6Al-4V alloy. The USRP-induced enhancement mechanism of the fatigue performance can be ascribed to the synergistic effect of the compressive residual stress, microstructure, work hardening, and improved surface quality. The best synergistic effect and, correspondingly, the greatest improvement in the fatigue performance were realized for the one-repeat USRP specimen. Display omitted •Fatigue limits are markedly improved via USRP with different processing numbers.•Fatigue enhancement degree decreases gradually with increasing processing number.•Moderate work hardening is more conducive to improving the fatigue performance.•The more degree of grain refinement does not mean the better fatigue performance.