Tobacco-specific N-nitrosamines (TSNAs) found in tobacco and its products are known to induce severe illnesses. Consequently, it is imperative to implement measures for diminishing TSNAs levels in tobacco as part of tobacco harm reduction strategies. Several bacteria have exhibited the capacity to break down TSNAs, although the efficiency of their degradation is frequently influenced by fermentation conditions. Therefore, optimizing the fermentation process is essential to improve the efficiency of the microbial degradation of TSNAs. However, the mechanisms involved in the microbial degradation of TSNAs remain unknown. In this context, the present study utilized a combined transcriptomics and proteomics analysis to reveal the mechanism underlying the degradation of TSNAs by Bacillus pumilus strain 05–5402. The TSNAs in flue-cured tobacco were degraded, and the fermentation process was optimized to determine the best conditions for the functioning of strain 05–5402. A TSNA degradation rate of 31.99% was reached at 28 °C and a water content of 100%. The analysis of both transcriptomics and proteomics data unveiled a range of mechanisms contributing to the degradation of TSNAs by strain 05-5402. Notably, amino acid biosynthesis, carbon metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, and the metabolism of various amino acids stood out as the most significant pathways. These findings can serve as a reference for the degradation of TSNAs using Bacillus pumilus fermentation at the industrial scale.