The microstructural transition and mechanical properties of Fe-Cr-2Ni-Mo-V steel were investigated in a carburizing process with carburizing at 935 °C, oil quenched, re-quenched at 880 °C,and low-temperature tempered at 180 °C. X-ray diffraction was employed for phase analysis and Ir Carbon-sulfur Spectrometer was used for carbon content detection. The morphologies of martensite were observed by scanning electron microscopy. The Vickers hardness was measured and the impact toughness was measured by means of Charpy’s V-notch tests. The results show that the carbon concentration and hardness gradients are established after carburizing process as a function of distance from the case to core. The carbon content gradient results in a gradient of martensitic microstructures, ranging from plate martensitic morphologies with large amounts of retained austenite in case to lath martensitic microstructures with essentially no retained austenite in core. The mechanical properties are significantly improved with adding re-quenching and low-temperature tempering. The case hardness, core hardness, tensile strength and impact energy are 851 HV, 402 HV, 1310 MPa and 92 J, respectively.