The effect of thermo-mechanical treatment on the microstructural evolution of low carbon micro-alloyed high strength steel was studied by combining prestrain with tempering (PST) in this paper. It was found that the prestrain causes the dislocation to plug up around the grain boundary and carbide, resulting in carbide boundary fragmentation. Moreover, it breaks the thermo-dynamic equilibrium between the matrix and carbide, induces the dissolution of carbon in the high energy state, and then changes the distribution of carbon in the matrix. In the subsequent tempering process, the precipitation regularity of carbide was changed, which promoted the precipitation carbide at low temperature. The influence of carbide precipitation on dislocation can be divided into two stages: the first stage was precipitation induced creep, which promoted stress relaxation; the second stage was precipitation pinning dislocation, which improved material strength and inhibited stress relaxation.