Abstract: Bifidobacterium exhibits distinct physiological subpopulations during cultivation, which influences its metabolic activity and enhancement of the number of viable bacteria. This study focused on Bifidobacterium longum subsp. infantis B8762 (B8762) to explore the growth performance differences among its cell subpopulations under various carbon sources. Flow cytometry sorting was employed to separate B8762 cells following high-density fermentation with lactose, sucrose, and maltose as carbon sources. The study compared and analyzed the differences in carbon metabolism and growth performance across these subpopulations to assess the impact of different carbon sources on bacterial cell viability. The results revealed that the number and turbidity of B8762 cells varied depending on the carbon sources (P<0.05), with distinct subpopulations showing different metabolic activities. Specifically, the activity of fructose-6-phosphate phosphoketolase (F6PPK) in the live cell subpopulations cultured with lactose, sucrose, and maltose was 1.47, 2.42, and 2.83 times higher, respectively, compared to the damaged cell subpopulation. After re-cultivation, the viable bacteria count in the sucrose-cultured live cell subpopulation reached (2.46±0.01)×10⁹ CFU/mL, significantly surpassing that of the other subpopulations (P<0.05). In conclusion, sucrose outperforms lactose and maltose as a carbon source in promoting cell activity, offering new insights and methods for optimizing the cultivation and preparation of highly active bacterial powders for dominant cell populations.