Effect of quenching process on the microstructure and mechanical properties of a kind of seamless tubes of steel 28CrMnMoV was investigated. Then, an investigation on the influence of two different quenching processes on the ductile-brittle transition behavior of this steel was undertaken. The ductile-brittle transition temperatures of the steel by two different quenching processes were also determined. The results show that a good combination of mechanical properties can be obtained through austenitizing experimental steel at 800 °C or 890 °C followed by tempering at 630 °C. Ductile-to-brittle transition temperature of 28CrMnMoV steel austenitized at 800 °C followed by tempering at 640 °C is about −73 °C, which is much lower than the value −37 °C when the steel was austenitized at 890°C and then tempered at 650 °C. This indicates that subcritical quenching process could decrease largely the ductile-to-brittle transition temperature of 28CrMnMoV steel.