Arsenic (As) contamination in paddy soils poses a severe threat to global food security, necessitating effective and sustainable soil management strategies. While microbial and biogeochemical processes largely govern As toxicity and mobility (e.g., As (Ⅲ) vs. As(Ⅴ)), the application of exogenous phytohormones as a practical agricultural management tool to regulate these processes remains underexplored. This study evaluates the efficacy of 2,4-epibrassinolide (EBL) application as a novel bioregulation strategy to promote As stabilization in paddy soils across varying contamination levels using a rice-cultivated microcosm system. Our results demonstrated that EBL intervention effectively improved soil micro-environments by elevating soil redox potential (Eh) and enriching organic carbon pools (TOC and DOC) via modulated root exudation. Microbiome and biochemical analyses revealed that EBL reshaped the soil microbial assembly and enhanced key soil enzymatic functions, significantly enriching functional taxa (e.g., Proteobacteria increased by 85.4–167.6% under high As stress) while suppressing stress-tolerant taxa (Firmicutes decreased by 69.0–80.4%). From a risk mitigation perspective, EBL achieved effective As stabilization by driving the conversion of highly mobile As(Ⅲ) to less toxic As(Ⅴ) and unextractable fractions, decreasing the proportion of soil As(Ⅲ) by 15% and increasing As(Ⅴ) by 14%. Consequently, this microbially and enzymatically driven As(Ⅴ) stabilization significantly reduced total As accumulation in rice roots and leaves by 10% and 30%, directly mitigating agricultural risk. Structural equation modeling confirmed that EBL governs this stabilization through the pathway of ”microbial community → soil physicochemical properties → As speciation”. Ultimately, this study validates EBL application as a promising, eco-friendly soil management intervention. By discussing the practical feasibility and dosage optimization for field-scale application, this work provides actionable insights for managing As-contaminated agricultural ecosystems and ensuring safe crop production.