The effects of vanadium content on the microstructural and mechanical properties of a newly developed hot-work die steel (MPS700V) at room and elevated temperatures have been investigated in this paper. It shows that the ultimate tensile strength (UTS) of MPS700V at room temperature and 700 °C are both strongly dependent on the V content. With the increase of V content from 0 to 1.2% V, The UTS at room temperature increases from 1127 MPa to 1442 MPa, and the UTS at 700 °C increases from 400 MPa to 550 MPa. The transmission electron microscope (TEM) shows that the addition of V is beneficial to increase the nucleation rate and volume fraction of MC nano-carbides during the tempering process, and the size of the MC nano-carbides decreases with the increase of V content. The addition of V also increases the thermal stability of MC nano-carbides at 700 °C, which can be related to the reducing the interface mismatch between MC and matrix. Furthermore, a transformation from Orowan bypass strengthening mechanism to shearing strengthening mechanism caused by the V addition is proposed based on the theoretical analysis and TEM observation.