${varepsilon}$-Poly-L-lysine (${varepsilon}$-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of Llysine, which is used as a safe food preservative. In this study, the effects of L-lysine and its isomer, D-lysine, on ${varepsilon}$-PL biosynthesis and their metabolites by the ${varepsilon}$-PLproducing strain Streptomyces ahygroscopicus GIM8 were determined. The results indicated that L-lysine added into the fermentation medium in the production phase mainly served as a precursor for ${varepsilon}$-PL biosynthesis during the flask culture phase, leading to greater ${varepsilon}$-PL production. At an optimum level of 3 mM L-lysine, a ${varepsilon}$-PL yield of 1.16 g/L was attained, with a 41.4% increment relative to the control of 0.78 g/L. Regarding D-lysine, the production of ${varepsilon}$-PL increased by increasing its concentrations up to 6 mM in the initial fermentation medium. Interestingly, ${varepsilon}$-PL production (1.20 g/L) with the addition of 3 mM D-lysine into the initial fermentation medium in flasks was higher than that of the initial addition of 3 mM L-lysine (1.06 g/L). The mechanism by which D-lysine improves ${varepsilon}$-PL biosynthesis involves its utilization that leads to greater biomass. After S. ahygroscopicus GIM8 was cultivated in the defined medium with L-lysine, several key metabolites, including 5-aminovalerate, pipecolate, and L-2-aminoadipate formed in the cells, whereas only L-2-aminoadipate was observed after D-lysine metabolism. This result indicates that Llysine and D-lysine undergo different metabolic pathways in the cells. Undoubtedly, the results of this study are expected to aid the understanding of ${varepsilon}$-PL biosynthesis and serve as reference for the formulation of an alternative approach to improve ${varepsilon}$-PL productivity using L-lysine as an additional substrate in the fermentation medium.