Autotrophic nitrogen-fixing hydrogen-oxidizing bacteria (NF-HOB) have shown promise for sustainable microbial protein (MP) production. This study enriched an efficient NF-HOB microbial community dominated by Xanthobacter in an airlift reactor, achieving a high biomass concentration of 4.8 g CDW/L and yield of 0.25 g CDW/L/d. The two-stage gas supply strategy implemented in the reactor improved NF-HOB growth, surpassing previous production rates. The produced MP had a superior essential amino acid profile to soybean meal and comparable to fish meal, making it a viable alternative for food and feed applications. The study emphasizes the importance of gas–liquid mass transfer in enhancing NF-HOB biomass yield and provides valuable insights into NF-HOB enrichment strategies for efficient microbial protein production.