Inflammatory bowel disease (IBD) is a chronic disease recognized as an autoimmune disorder. The purpose of this is to determine the impact of the E. coli strain Nissle 1917 (EcN) and its outer membrane vesicles (OMVs) on cytokine gene expression in an IBD cell line model (HT29) in the hope that these effects will guide the development of interventional therapeutic strategies for IBD. First, the cell viability of HT-29 cells treated with EcN and OMVs was evaluated using the MTT assay. Then, inflamed HT29 cells were inoculated with EcN bacteria (multiplicity of infection (MOI) 10, 100), and extracted OMVs (10, 50 mug/ml). Subsequently, cells were harvested for RNA extraction; followed by expression analysis using real-time PCR and also the evaluation of cytokines by ELISA assay. TNF-alpha, IL-12, and IL-1beta mRNA expression was decreased in EcN bacteria (MOI 10 and 100) and OMV 10 mug/ml group. IL-6 mRNA increased in the MOI 100 of ECN group but decreased in OMV mug/ml, despite elevated IL-6 protein levels in the OMV 50 mug/ml. Notably, no detectable secretion of IL-12, TNF-alpha, or IL-1beta was observed in ELISA assays, suggesting post-transcriptional regulation or low sensitivity. The findings revealed that EcN (MOI 10 and 100) enhanced anti-inflammatory IL-10 production, whereas OMV 10 mug/ml showed superior suppression on mRNA level compared to OMV 50 mug/ml.