In order to study the effect of initial surface roughness on water droplet erosion resistance of last stage blade substrate of steam turbine, eight 17-4PH samples were grounded and velvet polished by different mesh metallographic sandpaper to establish sample with different initial surface roughness. The water droplet erosion experiments were carried out in the highspeed jet water erosion experiment system, and the mass and micro-morphology of each sample were measured by using precision electronic balance and ultra-depth of field microscope respectively at each experimental stage, and the measurement of water erosion trace width and maximum water erosion depth were also completed at the same time. On the basis of experiments, LS-DYNA was used for numerical simulation to verify the reliability of experimental results again. Results show that the smoother the initial surface of sample, then the smaller the mass loss, the stronger its water erosion resistance. On the contrary, the rougher the initial surface of sample, the more severe the surface irregularity, the more times the water droplets concentrated at the lowest point of pit when water droplets flow laterally after impact is completed, thus accelerating the formation of initial crack and lateral expansion, the poorer the water erosion resistance of sample. At same water erosion time, the smoother the sample surface, the later the complete erosion trace appear, the narrower the water erosion trace width. However, the maximum water erosion depth of sample is not affected by the initial surface roughness. The numerical simulation results are in good agreement with the experimental results.