Abstract: To elucidate the mechanism of microbial inhibition on the formation of tobacco-specific nitrosamines (TSNAs), Bacillus flexus Ni3, a high-temperature-resistant strain capable of degrading nicotine with high efficiency, was isolated from cigar tobacco using the major precursors of TSNAs as the substrate. The inhibitory ability of strain Ni3 on TSNAs in cigar tobacco leaves was evaluated through shake flask culture experiments and simulated agricultural stack fermentation. The key genes involved in the degradation of TSNAs in strain Ni3 were identified by whole genome sequencing. The results showed that: 1) The nicotine degradation rate of strain Ni3 was 56% at 72 h in the nicotine fermentation medium with an initial nicotine mass concentration of 1.5 g/L. When the initial concentration of TSNAs was 10 mg/L, the degradation rates of NNN, NNK, NAT and NAB were 23%, 64%, 26%, and 27%, respectively. 2) Compared with the control group, the accumulation amounts of NNN, NNK, NAT and NAB on the 9th day of fermentation with Ni3 bacterial suspension decreased by 41%, 53%, 53% and 53%, respectively, and the total TSNAs decreased by 46%. 3) The whole genome sequencing analysis showed that there were 7 genes of strain Ni3 that might be involved in the degradation of TSNAs precursors. Therefore, B. flexus Ni3 has greater application potential in inhibiting the formation of TSNAs during the fermentation of cigar tobacco leaves.