Beta-cypermethrin (β-CY) residues in food are an important threat to human health. Microorganisms can degrade β-CY residues during fermentation of fruits and vegetables, while the mechanism is not clear. In this study, a comprehensively investigate of the degradation mechanism of β-CY in a food microorganism was conducted based on proteomics analysis. The β-CY degradation bacteria Gordonia alkanivorans GH-1 was derived from fermented Pixian Doubanjiang. Its crude enzyme extract could degrade 77.11% of β-CY at a concentration of 45 mg/L within 24 h. Proteomics analysis revealed that the ester bond of β-CY is broken under the action of esterase to produce 3-phenoxy benzoic acid, which was further degraded by oxidoreductase and aromatic degrading enzyme. The up-regulation expression of oxidoreductase and esterase was confirmed by transcriptome and quantitative reverse transcription PCR. Meanwhile, the expression of esterase Est280 in Escherichia coli BL21 (DE3) resulted in a 48.43% enhancement in the degradation efficiency of β-CY, which confirmed that this enzyme was the key enzyme in the process of β-CY degradation. This study reveals the degradation mechanism of β-CY by microorganisms during food fermentation, providing a theoretical basis for the application of food microorganisms in β-CY residues.