Electrocatalytic nitrogen reduction is a challenging process that requires achieving high ammonia yield rate and reasonable faradaic efficiency. To address this issue, this study developed a catalyst by in situ anchoring interfacial intergrown ultrafine MoO ₂ nanograins on N-doped carbon fibers. By optimizing the thermal treatment conditions, an abundant number of grain boundaries were generated between MoO ₂ nanograins, which led to an increased fraction of oxygen vacancies. This, in turn, improved the transfer of electrons, resulting in the creation of highly active reactive sites and efficient nitrogen trapping. The resulting optimal catalyst, MoO ₂ /C ₇₀₀ , outperformed commercial MoO ₂ and state-of-the-art N ₂ reduction catalysts, with NH ₃ yield and Faradic efficiency of 173.7 μg h ⁻¹ mg ⁻¹ _cat and 27.6%, respectively, under − 0.7 V vs. RHE in 1 M KOH electrolyte. In situ X-ray photoelectron spectroscopy characterization and density functional theory calculation validated the electronic structure effect and advantage of N ₂ adsorption over oxygen vacancy, revealing the dominant interplay of N ₂ and oxygen vacancy and generating electronic transfer between nitrogen and Mo(IV). The study also unveiled the origin of improved activity by correlating with the interfacial effect, demonstrating the big potential for practical N ₂ reduction applications as the obtained optimal catalyst exhibited appreciable catalytic stability during 60 h of continuous electrolysis. This work demonstrates the feasibility of enhancing electrocatalytic nitrogen reduction by engineering grain boundaries to promote oxygen vacancies, offering a promising avenue for efficient and sustainable ammonia production.