Abstract: On the basis of chemical composition of 4Cr5Mo2V steel, test steels with vanadium mass fractions of 0.15%, 0.55% and 1.25% were prepared, and then vacuum quenched at 1 040℃ and tempered at different temperatures (540, 620℃). The effect of vanadium content on the microstructure, hardness and impact toughness of the test steel was investigated. The results show that with the increase of vanadium content, the grains in all the tempered test steels were refined, and the number of precipitated VC-type carbides increased; the grain refinement and precipitation strengthening effects were enhanced, resulting in the increased hardness. The number of precipitated carbides after tempering at 650℃ was larger, leading to the softening of the matrix, therefore the hardness decreased compared with that tempered at 540℃. After tempering at 540℃, the test steel with vanadium content of 0.15% and 0.55% had ductile fracture, and the latter had better impact toughness, whose impact absorbing energy reached 265 J; when the vanadium content increased to 1.25%, the test steel had brittle fracture, and the impact absorbing energy was only 26 J. After tempering at 620℃, ductile fracture occurred in all three test steels, and the impact absorbing energy was greater than 200 J.