Abstract:[Background] As one of the major fungal diseases in cucumber production, powdery mildew occurs widely in the world, resulting in declined cucumber quality and yield and causing serious economic losses. [Objective] To isolate and identify the original strains for biocontrol against cucumber powdery mildew and to verify their biocontrol and plant growth-promoting effects. [Methods] Multiple methods including plate screening, 16S rRNA gene sequencing, phylogenetic analysis, whole genome sequencing, secondary metabolite analysis, and morphological characterization were employed to isolate, identify, and characterize the strains. [Results] An antagonistic bacterium S1 was obtained from the rhizosphere soil of cucumber with Bipolaris maydis as the indicator strain, and its growth-promoting effect on cucumber seedlings and control effect on powdery mildew were examined by pot experiments. The blast result of 16S rRNA gene sequences showed that this strain had a nucleotide identity of 100% compared with Bacillus subtilis KZH-E3. The whole genome sequencing combined with the phylogenetic analysis eventually identified the strain as B. subtilis. The pot experiments in a greenhouse proved that S1 had a significant biocontrol effect on cucumber powdery mildew. [Conclusion] B. subtilis S1 has obvious disease-preventing and growth-promoting effects on cucumber seedlings, having important research significance.