Tryptophan is an essential amino acid required for tumor cell growth and is also the precursor to kynurenine, an immunosuppressive molecule that plays a role in limiting anticancer immunity. Tryptophanase (TNase) is an enzyme expressed by different bacterial species that converts tryptophan into indole, pyruvate and ammonia, but is absent in theSalmonellastrain VNP20009 that has been used as a therapeutic delivery vector. We cloned theEscherichia coliTNase operontnaCABinto the VNP20009 (VNP20009-tnaCAB), and were able to detect linear production of indole over time, using Kovács reagent. In order to conduct further experiments using the whole bacteria, we added the antibiotic gentamicin to stop bacterial replication. Using a fixed number of bacteria, we found that there was no significant effect of gentamicin on stationary phase VNP20009-tnaCABupon their ability to convert tryptophan to indole over time. We developed a procedure to extract indole from media while retaining tryptophan, and were able to measure tryptophan spectrophotometrically after exposure to gentamicin-inactivated whole bacterial cells. Using the tryptophan concentration equivalent to that present in DMEM cell culture media, a fixed number of bacteria were able to deplete 93.9% of the tryptophan in the culture media in 4 h. In VNP20009-tnaCABdepleted tissue culture media, MDA-MB-468 triple negative breast cancer cells were unable to divide, while those treated with media exposed only to VNP20009 continued cell division. Re-addition of tryptophan to conditioned culture media restored tumor cell growth. Treatment of tumor cells with molar equivalents of the TNase products indole, pyruvate and ammonia only caused a slight increase in tumor cell growth. Using an ELISA assay, we confirmed that TNase depletion of tryptophan also limits the production of immunosuppressive kynurenine in IFNγ-stimulated MDA-MB-468 cancer cells. Our results demonstrate thatSalmonellaVNP20009 expressing TNase has improved potential to stop tumor cell growth and reverse immunosuppression.