The high cycle fatigue (HCF) property and microstructure of a dissimilar metal welded joint (DMWJ) for ultra-supercritical steam turbine rotor were systematically investigated in this paper. The DMWJ was fabricated using narrow gap submerged arc welding (NG-SAW) technique with buttering layer. Conditional fatigue strength of 30Cr1Mo1V (BM-1), buttering layer (BL), weld metal (WM) and 30Cr2Ni4MoV (BM-2) based on S-N curve was obtained by HCF tests at room temperature. The microstructure and second-phase carbide as well as fracture appearance of BM-1, BL, WM and BM-2 were characterized by optical microscopy (OM), scanning electron microscopy (SEM) and transmission electron microscope (TEM). The results show that the BL has lower fatigue strength, which are related to more content of the soft ferrite and less content of the hard carbide. The acicular ferrite represents better fatigue properties than massive ferrite due to barrier effect. Additionally, dislocation density sharply increases near the carbides, grain boundaries, and lath structures resulting in dislocation tangle after HCF test, which helps to improve the fatigue strength of the welded joint. The carbide precipitated, aggregated, and grown in the grain boundaries promoted crack nucleation, reduced the crack propagation rate and affected the crack propagation path.