Abstract Immune checkpoint inhibitors (ICI) targeting PD-1, PD-L1, or CTLA-4 have shown remarkable therapeutic outcomes in various cancers. However, the therapeutic efficacy of ICI-based immunotherapy in microsatellite stable (MSS) colorectal cancer (CRC) has been extremely limited. Recent studies strikingly revealed that the human gut microbiome controls the efficacy of immune checkpoint blockades. Yet, the effect of the gut microbiome on reinforcing the ICI efficacy in MSS tumors in CRC is unknown. Here, we suggest a microengineered CRC Chip that recapitulates the tumor microenvironment of individual CRC patients by introducing biopsy-derived colonic organoid epithelium. We have built a three-dimensional (3D) lumen-capillary interface under peristalsis-like motions and flow in an oxygen-controlled microenvironment. We leverage this pathomimetic CRC Chip to recapitulate the patient-specific tumor-microbiome-immune axis to demonstrate microbial immunomodulation on the tumor cells and cytotoxic tumor immunity. We co-cultured various human intestinal bacteria in a CRC Chip to assess the immunomodulatory contribution to the tumor epithelium. We found that living Bifidobacterium sp. significantly reduces the PD-L1 on the tumor surface, followed by elevated anti-tumor activity after CD8 T cell-mediated cytotoxic interactions. We envision that our CRC Chip model can potentially reduce the knowledge gap between the bench and bedside. Furthermore, our innovative CRC Chip model may potentially offer a platform to validate the “Druggable microbiome” and the utility of the patient’s avatar model for Precision Medicine. Citation Format: Yong Cheol Shin, Alexander Wu, Soyoun Min, Daesung Kim, Woojung Shin, S. Gail Eckhardt, R. Y. Declan Fleming, Hyun Jung Kim. Microbial immunomodulation for enhancing immunotherapeutic efficacy in a patient-specific colorectal cancer chip [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3862.