[Objective] To explain the reason of the improvement of ε-poly-L-lysine (ε-PL) in Streptomyces by genome shuffling (GS) through the analysis of ε-PL metabolic flux distribution between the parent strains and the high yield strain. [Methods] Glucose tolerant Streptomyces sp. AS32 and ε-PL tolerant Streptomyces albulus F15 were selected as parent strains for GS. Three rounds of GS were carried out and a higher ε-PL producing shuffled strain Streptomyces sp. AF3-44 was obtained. The ε-PL synthetic network was constructed by metabolic flux analysis method and the metabolic fluxes among the above strains were compared. [Results] Shuffled strain, AF3-44, had a flask yield of 3.1 g/L which was increased by 34% and 29% compared with those of parent strains AS32 and F15 respectively. The fluxes to the tricarboxylic acid cycle (TCA) were highest in AS32, whereas and the fluxes to the pentose phosphate pathway (PPP) are highest in F15. AF3-44 had highest fluxes from oxaloacetic acid to aspartic acid and ε-PL synthesis, while it had moderate TCA and PPP fluxes, where the flux to isocitrate in TCA was 77% and 116% of those in AS32 and F15 respectively, and the flux to ribulose-5-phosphate was 149% and 92% of those in AS32 and F15 respectively. [Conclusion] Genome shuffling led to increase of flux to the precursor lysine and ε-PL, and the redistribution of metabolic fluxes in PPP and TCA which contributed to the improvement of ε-PL in the shuffled strain.