Limited information is available on the phenotypic characteristics of Listeriamonocytogenes that may affect its virulence of L.monocytogenes. The study focused on comparing the differences between two strains of L. monocytogenes, one hemolytic (LLO+) and one non-hemolytic (tLLO). Using Phenotype Microarray (PM) technology, carbon, nitrogen, and phosphorus-sulfur source utilization by the strains was performed and the KEGG pathway involvement in substrate utilization was analyzed. Of the 285 substrates screened, the results suggested that d-gluconic acid and l-serine may play a role in L. monocytogenes hemolytic activity towards LLO production by generating pyruvate as an intermediate and amino acid biosynthesis, which may play an important role in virulence protein translation. d-gluconic acid and l-serine showed no effect on the tLLO strain in PM analysis and validation tests. Notably, the scope of this study was limited to these two strains of L. monocytogenes. While these findings provide valuable insights into the metabolic differences between hemolytic and non-hemolytic strains, further research is necessary to expand this understanding to a broader range of strains and explore additional factors that may influence LLO functionality. •There are metabolism differences between L. monocytogenes producing active LLO and truncated LLO.•l-serine and d-gluconic acid were identified as key metabolites that may affect hemolytic activity.•l-serine and d-gluconic acid metabolism generates PRPP and pyruvate as intermediates, respectively.•The metabolism of the intermediates synthesized amino acids may be involved in LLO formation.