Reduction of MoO3 in extreme reducing condition is a way to achieve Mo metal. However, effect of less extreme reductive-annealing, where it allows to keep the crystal structure, on physical and chemical properties of MoO3 has not been well-studied. In this work, we studied the evolution of Mo valence state during reductive annealing and its effect on high temperature transport. We found the formation of oxygen vacancies on surface of MoO3 single crystals at the low temperature, which is evidenced by increase of Mo5+ and color change. In addition, formation of Mo4+ was at the elevated temperature. For understanding the relation between bulk conductivity and Mo valence state, real-time impedance spectroscopy is employed. Use of two different gases makes it possible to distinguish impedance responses of MoO3 from those of reduced MoO3-x. Also, from time-dependent impedance measurements, we observed the evolution of transport behaviour by evolution of Mo valence state.