Abstract: The surface and section crack morphology, microstructure and hardness changes of 4Cr5MoV hot work die steel containing different mass fraction (1.8%, 3.2%) of Mo were analyzed by self-constrained cold and thermal fatigue test from room temperature to 600℃. The effect of Mo content on cold and thermal fatigue properties of hot work die steel was studied. The results show that after 1 000 cold and hot fatigue cycles, the cracks on the surface of test steel containing 3.1wt% Mo were thin and sparse, and the crack number was significantly less than that of the test steel containing 1.8wt% Mo; the test steel containing 3.1wt% Mo had higher temper softening resistance, and high surface hardness could delay the initiation of cold and thermal fatigue cracks in the die steel. After 2 000 cold and thermal fatigue cycles, the crack propagation depth of the test steel containing 3.1wt% Mo was greater than that of the test steel containing 1.8wt% Mo, indicating the crack propagation rate was larger, which was related to the low impact toughness at room temperature and coarsening of a large amount of carbides of the test steel containing 3.1wt% Mo.