This study aimed to assess the applicability of freeze-dried E. faecium NRRL B-2354 (FDE) to evaluate the inactivation efficiency of radio-frequency (RF) pasteurization on wheat flour. FDE was prepared by adding equal volume of 10% skim milk solution as a protectant. Microstructure of FDE was evaluated by scanning and transmission electron microscopy. Thermal resistance parameters of FDE in wheat flour were determined at 75 degrees C, 80 degrees C, and 85 degrees C. A 6 kW, 27.12 MHz RF heating system was used to rapidly pasteurize 3.0 kg wheat flour at 75 degrees C, 80 degrees C, and 85 degrees C, respectively. Population reduction of FDE in the geometric center of flour samples were compared with survivor counts and predicted model. Flour samples were equilibrated to water activity 0.45 measured at 25 degrees C. The results showed that FDE protected by skim milk were well maintained with repeatable D-values D-75 degrees C = 14.27 +/- 0.85 min, D-80 degrees C = 5.92 +/- 0.39 min, and D-85 degrees C = 2,79 +/- 0.15 min, and z value of 13.1 degrees C. RF treatment at 75 degrees C, 80 degrees C, and 85 degrees C for 10 min achieved 1.0-, 2.5-, and 4.9-log reduction of FDE, respectively. In RF pasteurization process, the dynamic population reduction of FDE can be predicted by a model based on F-value. RF treatment provides an effective and rapid approach to inactivate target microorganism in flour samples.