The synthesis of NH3 from N2 and H2 is a significant chemical process, because essential products such as fertilizers and industrial chemicals can be obtained from NH3. In the catalytic synthesis of NH3, N2 activation is generally considered a kinetically relevant step (KRS). Therefore, advanced heterogeneous catalysts that promote N2 conversion have been extensively investigated. In this study, the roles of O and N vacancies (Ov and Nv) of Mo oxide formed during reduction were kinetically evaluated and compared using structural characterization, isotope experiments, kinetic studies, and density functional theory calculations. On the Mo oxide catalyst with Ov, NH3 was formed by a dissociative mechanism, and the surface reaction involving the adsorbed N* and H* was considered the KRS. However, on the catalyst with Nv, NH3 was formed by an associative mechanism, and N–N dissociation of N2H2* species by H* to produce NH3 was considered the KRS. The catalyst with Ov resulted in an NH3 synthesis rate of 5.6 mmol gMo−1 h−1 under relatively mild conditions (1.0 MPa and 400 °C), thus outperforming the catalyst with Nv (0.3 mmol gMo−1 h−1). These results provide insight into the importance of anionic vacancies in reducible metal oxides for NH3 synthesis.