Abstract: pH control was a key regulatory strategy in the high-density cultivation of Bifidobacteria. However, research into the mechanisms by which pH regulates cell division and proliferation remains limited. In this study, Bifidobacterium breve B2798 was used as the model organism. Cells were labeled with the fluorescent proliferation probe Carboxyfluorescein diacetate succinimidylester (CFDA-SE), and flow cytometry was utilized to monitor bacterial division dynamics under varying fermentation pH conditions. Quantitative real-time PCR (qPCR) was applied to measure the expression levels of key genes involved in cell division, aiming to investigate the influence of fermentation pH on its division cycle. The results showed that at a fermentation pH of 5.30, cells underwent up to 7 division cycles, a higher number than observed at other pH levels. The viable cell count at this pH was (1.39±0.04)×10¹⁰ CFU/mL, significantly higher than that at other pH (P<0.05), indicating that cells not only divide actively but also maintain high viability. At pH5.30, the expression of division-related genes ftsZ and sepF was upregulated by 61.86% and 131.36%, respectively, compared to pH6.30 (P<0.05), and by 78.13% and 80.31%, respectively, compared to pH4.30 (P<0.05). In conclusion, fermentation pH regulates cell division by influencing viable cell counts and regulating the expression of division-related genes, collectively creating an optimal environment for rapid cellular proliferation. This mechanistic insight provides theoretical support for precision fermentation in achieving high-density cultivation and yield optimizatio