The microstructure, mechanical properties, and heat treatment process relationships of 45MnVS non-tempered steel were investigated. Different grain sizes and tissue ratios were obtained after heating and holding at 950, 1050, and 1150 °C for 30 min, respectively, and cooling at different cooling conditions and cooling rates. As the austenitizing temperature increased, the grain size and the elongation and section shrinkage of the experimental steels decreased. At the same time, the content of ferrite and pearlite in the tissue gradually decreases, the content of sorbite and troostite rises, and the strength and hardness of the experimental steel increase. With the increase of cold speed, ferrite’s content decreases, sorbite and troostite’s content increases, and strength and hardness increase. The original austenite grain size also influences the tissue ratio. As the austenite grain refines, the ferrite transformation temperature increases during cooling and the ferrite content in the tissue rises after cooling.