Refined microstructures achieved by cyclic heat treatment significantly contribute to improving the wear resistance of steels. To acquire the refined microstructures of 65Mn low-alloy steel, first, the specimens were solid solution-treated; then, they were subjected to cyclic heat treatment at cyclic quenching temperatures of 790–870 °C and quenching times of 1–4 with a fixed holding time of 5 min. The mechanical properties of 65Mn low-alloy steel in terms of hardness, tensile strength, elongation and wear resistance were characterized. Afterwards, the effect of cyclic heat treatment on microstructure evolution and the relationships between grain refinement and mechanical properties’ improvement were discussed. The results show that the average grain size firstly decreased and then increased with the increase in the quenching temperature. Hardness increased with grain refinement when the temperature was lower than 830 °C. Once the temperature exceeded 830 °C, hardness increased with the temperature increase owing to the enrichment of carbon content in the martensite. With the increase in cyclic quenching times, hardness continuously increased with grain refinement strengthening. In addition, both tensile strength and elongation could be significantly improved through grain refinement. The relationships among wear loss, hardness and average grain size showed that wear resistance was affected by the synthesis reaction of grain refinement and hardness. Higher hardness and refined grain size contributed to improving the wear resistance of 65Mn low-alloy steel.