High-temperature fatigue test was performed to study the effects of laser peening (LP) on the fatigue behavior of Inconel 718 nickel-based alloy at high temperature. Compressive residual stress (CRS) induced by LP was investigated at both room and high temperature. Microstructural evolution was also studied before and after LP process. The macro- and micro-fracture morphologies in fatigue crack initiation (FCI) region, fatigue crack growth (FCG) region and finial rupture (FR) region of LPed samples with different laser power densities at different temperatures were systematically analyzed. The results showed that LP process can enhance the fatigue life of IN718 nickel-based alloy at both room and high temperature. Excessively high temperature (e.g. 800 °C) may result in a sudden decrease in the fatigue life of LPed samples due to the weakening of grain boundaries and oxidation damage. The losing of coherent strengthen between γ″ phase and matrix was another reason for the rapid reducing of fatigue life. Compared to the untreated samples, the crack source transferred from the surface to deeper layer indicated that LP can retard the crack initiation on the metal surface, which is conducive to enhance the final fatigue life. Some direct evidence, such as the decreased distance between the parallel fatigue striations, the tortuous crack growth path in the LPed specimen confirmed the retarding effects of LP on the FCG. The mechanism of microcracking and the dynamic plastic behavior of LPed specimens during high-temperature fatigue test of IN718 nickel-based alloy were also discussed.