Abstract: The development of antitumor live microbial agents represents an emerging frontier in cancer immunotherapy. Attenuated Salmonella VNP20009 stands as one of the most extensively studied oncolytic bacterial strains, demonstrating remarkable tumor-targeting specificity with 10³–10⁴-fold higher distribution titers in tumor tissues compared to normal tissues, alongside significant antitumor efficacy. However, the molecular mechanisms underlying its tumor-targeting properties and antitumor activity remain elusive, hindering further strain optimization. As aromatic amino acids serve as essential nutrients for bacterial growth, their biosynthesis in Salmonella is primarily regulated by the aroC gene. To investigate the role of aromatic amino acid biosynthesis in VNP20009's antitumor activity, we constructed an aroC-deficient strain (VNP ΔaroC) using CRISPR/Cas9 technology. Systematic evaluations were performed to assess: ① tolerance to extracellular stresses (pH, H₂O₂) in vitro; ② invasive capacity against B16F10 melanoma cells; ③ proliferative capacity within tumor cells; ④ antitumor efficacy and biodistribution in B16F10 tumor-bearing mice. Experimental results revealed that VNP ΔaroC exhibited comparable growth profiles to wild-type VNP20009 under varying pH and H₂O₂ conditions. However, its colony growth rate on solid medium under identical pH and H₂O₂ conditions was significantly reduced relative to VNP20009. Furthermore, the invasive and proliferative capacities against B16F10 melanoma cells were markedly diminished compared to the wild-type strain. In murine models, VNP ΔaroC demonstrated reduced systemic toxicity but concurrently showed attenuated antitumor efficacy and tumor-targeting specificity. These findings establish aroC as a critical gene governing both therapeutic potency and tumor-targeting capability of VNP20009. This study provides valuable insights for further synthetic biological engineering of oncolytic bacterial strains. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Nanjing University (approval No: IACUC-2003167).