The deformation behavior of 49MnVS3 non-quenched and tempered steel was studied using isothermal compression tests at the temperature range of 750–1000 °C and the strain rate range of 0.1–50 s−1 on a Gleeble-3500 thermal mechanical simulator. The results indicated that the flow stress increases significantly with decreasing temperature and increasing strain rate. Under the whole deformation conditions, the dependence of flow stress on deformation temperature and strain rate was analyzed by hyperbolic sine equation. Besides, the hot deformation activation energy and stress exponent were calculated to be 323.56 kJ/mol and 6.99, respectively. In addition, the processing map based on dynamic material model was established, and the optimum processing condition of the 49MnVS3 non-quenched and tempered steel can be determined to be at the temperature range of 809–850 °C and strain rate of 36.6 s−1–50 s−1.