Tumor necrosis factor (TNFα) is a proinflammatory cytokine involved in the pathogenesis of inflammatory bowel disease (IBD). Although TNFα has been extensively targeted using systemic drugs, the use of antisense and small interfering RNA (siRNA) to drive down its expression at the site of inflammation should provide important advantages. In this study, native and chemically modified siRNA against TNFα was developed and characterized using a murine model of IBD. siRNA with 2′-O-methyl and propanediol modifications (siTNF-OMe-P) were resistant to nuclease degradation and provided better silencing efficacy in vitro as compared to unmodified siRNA. Every modification reduced nonspecific Toll-like receptor (TLR)-mediated immunomodulation in human peripheral blood mononuclear cells (PBMC) cells. Intrarectal administration of siTNF-OMe-P significantly ameliorated the clinical endpoints and histopathological severity in 5% dextran sulphate sodium (DSS)-treated mice as compared to unmodified and other chemically modified siRNAs. Differential gene expression assessed in siTNF-OMe-P-treated animals correlated with improved colon integrity and reduced TLR activation as compared to all treatment groups. All in all, this study demonstrates that propanediol and 2′-O-methyl modifications have profound functional consequences for siRNA efficacy in vivo. Consequently, this strategy has potential implications for therapeutic intervention in IBD and other diseases. © The American Society of Gene & Cell Therapy.