The relationship between microstructure and flow behaviour has attracted attention from many researchers for the past decades, whilst the influences of dislocation and recrystallization on flow stress have not been well understood, which led to failure in flow stress prediction at high temperature compressions. In this work, we tried to provide a novel explanation of the relationship between microstructure evolutions and flow behaviour, and the influence of dislocation and recrystallization on flow stress was investigated. A dislocation based flow stress model was proposed and applied for 300M steel at the strain rate of 0.01–10 s−1 and the temperature of 950–1150 °C. Results showed the established model could predict the flow stress both at constant strain rate conditions and at variable strain rate conditions. The present investigation is helpful to a better understanding of hardening and softening mechanisms in hot compression of 300M steel.