Fusobacterium nucleatum (Fn) is significantly associated with poor prognosis in colorectal carcinoma (CRC), however, mechanisms of Fn in DNA mismatch repair (MMR) and microsatellite instability (MSI) in CRC have not been fully elucidated. Clinical samples are collected to analyze the relationship between Fn abundance and microsatellite stability. Tumor cells are treated with Fn to detect the expression of proteins related to toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (Myd88), mutS homolog 6 (MSH6), and nuclear factor-κB (NF-κB) signaling pathways, respectively. Combined with the prediction results from TargetScan, the regulatory role of microRNA upstream of MSH6 is demonstrated. The effect of this regulatory axis on CRC development is demonstrated using a nude mouse tumor model. Compared with microsatellite stability (MSS)-type CRC patients, MSI-type showed higher Fn abundance. Fn treatment of CRC cells activated TLR4/Myd88/NF-κB signaling pathway, transcriptionally activating miRNA-155-5p expression, thereby negatively regulating MSH6. Fn treatment accelerated the malignant progression of CRC in mice, and this process is inhibited by miRNA-155-5p antagomir. Fn in CRC upregulated miRNA-155-5p by activating TLR4/NF-κB signaling to inhibit MSH6, and this regulatory pathway may affect MSS of cancer cells.