Abstract: This study aimed to explore the potential application of probiotics for the safe removal of zearalenone (ZEN) from feed by screening and validating the ZEN transformation capability of a soil-derived Bacillus subtilis strain 23830. Critical fermentation parameters were systematically optimized to maximize ZEN removal efficiency. The structural characterization and in vitro toxicity profile of biotransformation products were analyzed using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) and HepG2 cytotoxicity assays. The strain was further evaluated for practical application through fermentation trials on corn by-products. Key findings revealed that ZEN-transforming proteases were predominantly localized in the intracellular fraction. Under optimal transformation conditions (2% inoculum 1×10⁷ CFU/mL, 37 ℃, pH 7.0, 12 h incubation), a ZEN removal rate of 60.47%±2.18% was achieved. Structural analysis identified zearalenone-14-sulfate (ZEN-14-Sulf) as the primary metabolite, which demonstrated significantly reduced cytotoxicity compared with the parent compound. In practical applications, the strain exhibited differential detoxification efficiencies across substrates: 15.67%±0.64% (corn germ meal), 19.22%±2.35% (corn gluten feed), 30.71%±0.79% (corn protein powder), and 45.44%±2.73% (corn slurry). The Bacillus subtilis strain 23830 characterized in this study represents a promising microbial candidate for feed detoxification, enabling both effective ZEN reduction and low-toxicity biotransformation. These findings advance the development of probiotic-based strategies for mycotoxin mitigation in agricultural systems.