Laser welding is used to weld 10Ni5CrMoV high strength steel at vacuum. The effects of ambient pressure on the microstructure of weld bead and mechanical properties are studied. The results show that laser welding at vacuum can improve the weld formation quality and increase penetration depth. The ambient pressure has little effect on microstructure of weld bead, while had obvious influence on the microstructure of heat-affected zone. Weld beads obtained at different ambient pressures are all composed of martensite. With the decrease of ambient pressure, carbides precipitate out in heat-affected zone gradually, the microstructure transforms from martensite to martensite with carbide and very few granular bainite, and a small amount of ferrite are obtained. The decrease of martensite content is the main factor of the microhardness decrease in vacuum environment. When the ambient pressure is 10 Pa, the weld hardness decreased by 6.2% compared with the atmosphere. The tensile samples are all broken at the base metal with obvious necking, and the fracture mode is ductile fracture.