Abstract: Objective To address problems of cold cracking, reheat cracking, low welding efficiency, and complex processing in the conventional narrow-gap submerged arc welding of 2.25Cr-1Mo-0.25V steel hydrogenation reactors, feasibility of applying vacuum electron beam welding to thick-section welding of this steel was investigated in the paper. Methods Vacuum electron beam welding experiments were carried out on 40 mm thick 2.25Cr-1Mo-0.25V steel plates, and the optimal welding parameters were determined by adjusting beam current. On this basis, microstructure evolution of welded joints in the as-welded condition and after simulated maximum post-weld heat treatment and simulated minimum post-weld heat treatment was analyzed, and room-temperature tensile properties, high-temperature tensile properties at 454 ℃, low-temperature impact toughness at −30 ℃, and transverse side-bend properties of welded joints were evaluated. Results The results showed that defect-free welded joints with a thickness of 40 mm could be obtained under the conditions of under-focus, welding speed of 200 mm/min, accelerating voltage of 85 kV, and beam current of 126 mA. After post-weld heat treatment, microstructure of base metal remained basically unchanged, martensite in heat-affected zone decomposed, and weld zone transformed into lath bainite and retained martensite. Conclusion Strength, low-temperature impact toughness, and side-bend properties of welded joints all meet the technical requirements, indicating that vacuum electron beam welding of 2.25Cr-1Mo-0.25V steel hydrogenation reactors has good engineering application prospects.