Abstract: Electron beam welding was carried out between 40 mm thick ferritic stainless steel plate and 16Mn steel plate by electron beam biased to the side of 16Mn steel, and the microstructure and impact toughness of different areas of the welded joints were investigated. The results show that the microstructure of the weld was mainly lath martensite; with increasing distance from the upper surface of the weld, the impact toughness of the weld decreased, and the fracture was characterized by quasi-cleavage and uncleavage. The microstructure of the heat affected zone at 16Mn steel side was mainly composed of acicular ferrite and feathery upper bainite; the maximum impact absorption energy was 113 J, and the impact toughness was the best; the fracture type was ductile and brittle mixed fracture. The microstructure of the heat affected zone at ferritic stainless steel side was mainly composed of ferrite, martensite and granular carbides; the impact absorption energy ranged from 6 J to 10 J, and the impact toughness was the worst; the fracture type was brittle fracture, which was related to the coarse ferrite grains and brittle carbides.