Deep cryogenic treatment (DCT) is believed to have superior performance in coordination with traditional heat treatment. In present work, the effect of DCT combined with inter-critical quenching treatment on the mechanical properties and microstructure of 30CrMnSi alloy structural steel was investigated. Before DCT employed, quenching treatment was conducted at different austenitizing temperatures (780 °C, 820 °C and 900 °C) to obtain diverse initial states of microstructure. The influence of different sequence between DCT and tempering was also studied. The results showed that compared to quenching-tempering (QT) treatment, the impact toughness of 30CrMnSi alloy austenitized at all the three temperatures was improved by conducting DCT after quenching and tempering (QTC). The strength and toughness were concurrently improved by quenching-tempering-cryogenic (QTC) treatment at the austenitizing temperature of 820 °C. Microstructural characterizations were carried out by optical microscopy (OM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and electron backscattered diffraction (EBSD). Strip ferrite and lath martensite were refined, as well as high-angle grain boundaries increased after 820-QTC. Deep cryogenic treatment has also promoted the dispersed precipitation of stable carbides in the boundaries of ferrite and martensite, through constricting the martensite lattice and increasing the nucleation energy under ultra-low temperature.