Aims <p>Associative nitrogen fixation represents a promising alternative to chemical fertilizers. However, diazotrophs that combine high nitrogen-fixation efficiency with strong rhizosphere colonization ability in cereal crops remain extremely scarce. This study aimed to isolate and characterize novel associative diazotrophs from the rhizosphere of white clover (<i>Trifolium repens</i> L.) and to evaluate their potential to promote plant growth.</p> Methods <p>The associative diazotrophic consortium was enriched from the white clover rhizosphere and screened for nitrogenase activity. The most efficient isolate, identified as a potentially novel species within the genus <i>Paenibacillus</i> (designated as strain SY6), was characterized through acetylene reduction assays, plant growth-related trait analysis, and whole-genome sequencing. Maize was used as a model crop to evaluate rhizosphere colonization and growth promotion under nitrogen-limited hydroponic conditions.</p> Results <p>The strain SY6 exhibited high nitrogenase activity (123.39&#xa0;nmol C₂H₄ h⁻¹&#xa0;mL⁻¹), significantly exceeding that of the reference strain <i>Klebsiella variicola</i> W12 (<i>P</i> &lt; 0.05) and reported associative diazotrophs. Genome analysis revealed complete molybdenum-dependent (<i>nif</i>) and vanadium-dependent (<i>vnf</i>) nitrogenase systems, an indole-3-pyruvic acid (IPyA) pathway for IAA biosynthesis, and multiple amino acid synthesis genes. Inoculation of maize with SY6 resulted in over 20% increase in biomass and a 16—30% elevation nitrogen content, demonstrating effective rhizosphere colonization and enhanced nitrogen assimilation.</p> Conclusions <p><i>Paenibacillus</i> sp. SY6 represents a potentially novel, highly efficient associative diazotroph with dual nitrogenase systems and phytohormone-producing capacity. Its robust cross-host colonization and significant plant growth promotion suggest strong potential as a microbial biofertilizer for sustainable agriculture.</p> Graphical abstract <p></p>