Aims <p>Soil heterotrophic ammonia-oxidizing bacteria (HAOB) play a key role in nitrogen cycling and plant growth, yet their contribution to cytokinin biosynthesis remains poorly understood.</p> Methods <p>We investigated the effects of two HAOB strains (S2_8_1 and A8) on <i>Lolium multiflorum</i> regrowth by conducting pot experiments, bacterial culture assays, and bioinformatics analysis. We assessed soil net nitrification, cytokinin production, and plant regrowth responses to determine the functional roles of these strains.</p> Results <p>Functional gene analysis revealed that S2_8_1 possesses the tRNA isopentenyl transferase (MiaA) for tRNA-dependent cytokinin biosynthesis, LONELY GUY (LOG) homologs for cytokinin activation, and CYP450-mediated heterotrophic nitrification, compensating for the absence of traditional isopentenyl transferase (IPT) and ammonia monooxygenase (AMO) pathways. Pot experiments showed that S2_8_1 inoculation increased soil NO₃⁻-N content by 18% and net nitrification by 21% compared to A8 by day 14. Enhanced nitrate availability stimulated cytokinin transport, leading to a 40% increase in leaf regrowth. Bacterial culture assays confirmed that S2_8_1 produced 2.53 times more cytokinins than A8, further supporting its role in cytokinin biosynthesis. This combined effect of improved nitrogen transformation and cytokinin production resulted in a 40% increase in clipped leaf biomass.</p> Conclusions <p>Our findings highlight the dual role of S2_8_1 in nitrogen cycling and phytohormone regulation, suggesting potential as a microbial biofertilizer. While S2_8_1 increases NO₃⁻ via heterotrophic nitrification, its benefit depends on synchrony with plant uptake. When timed with crop demand, this may improve nitrogen use and reduce fertilizer input, though leaching or gas losses remain risks under poor management.</p>