Abstract: Thermal fatigue cracking is the main failure mode of hot work die steel during die casting and hot forging. Thermal fatigue cracking accounts for a large proportion of mold failures and seriously affects the service life of the mold. Because of the high maintenance and replacement costs, thermal fatigue failure will cause substantial financial losses to the enterprise. Therefore, analyzing the fatigue behavior of hot work die steel at high temperatures is of significance in scientific research and engineering applications. H13 hot work die steel is widely used in die casting and hot forging because of its excellent high-temperature performance and toughness. In this study, a 600 ℃ isothermal fatigue test was conducted on H13 hot work die steel samples. The effect of three different strain amplitudes of 0.7%, 0.9%, and 1.1% on the isothermal fatigue behavior was analyzed using a micro Vickers hardness tester, metallographic microscope, microscope with a superwide depth of field, and scanning electron microscope. Results show that the stress–strain hysteresis loop is symmetric. The larger the strain amplitude is, the larger the area of the hysteresis loop. H13 hot work die steel exhibits the cyclic softening behavior during the experiment. The larger the strain amplitude, shorter is the fatigue life. The fatigue life of the sample with the strain amplitude of 1.1% is approximately 61.2% of that of the sample with the strain amplitude of 0.7%. The increase in the strain amplitude promotes the initiation and propagation of cracks, and the propagation of cracks on the sample with the strain amplitude of 1.1% is the most obvious. Under high-temperature and non-vacuum experimental conditions, oxide on the surface of the material promotes crack growth. The microstructure of the sample under isothermal fatigue grows and coarsens. The large strain amplitude not only supports carbide precipitation but also accelerates cyclic softening of the material. The microhardness of samples with strain amplitude is lower than that of samples without strain amplitude.