Herein, the microstructure evolution and mechanical properties of the 40CrNiMo steel treated by different austempering temperatures are investigated. With the decrease in austempering temperatures from 270 to 180 °C, the volume fraction of bainite decreases, and the martensite fraction increases. The austenite content is about 8% at 270 °C, and then increases over 12% at 240≈180 °C. The mean grain size decreases from 270 to 180 °C, which is related to the reason that the development of bainite/austenite interface is restricted for the martensite formation. The yield strength increases from 1153 to 1323 MPa by refinement hardening and dislocation hardening, and ultimate tensile strength increases from 1193 to 1731 MPa at from 270 to 180 °C. The elongation is about 13% at 210≈270 °C, and then decreases at 180 °C.