Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) the etiology of which has not yet been fully clarified. Cytokine interleukin-10 (IL-10) plays a central role in downregulating inflammatory cascade in UC and is likely a candidate for therapeutic intervention. However, its intravenous administration is costly and inconvenient. Therefore, we established a novel IL-10 delivery system by transforming a hIL-10-containing plasmid into B. longum (BL-hIL-10) and investigated its effects on 5% dextran sulfate sodium (DSS)-induced ulcerative colitis in mice and the possible underlying mechanism. Our results show that (1) hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after L-arabinose induction in vitro as examined by Western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR; (2) addition of BL-hIL-10 culture supernatant had no cytotoxic effect and morphological alteration, but significantly inhibited the enhancement of proinflammatory cytokines by lipopolysaccharide (LPS) in THP-1 cells; (3) oral administration of BL-hIL-10 alleviated colitis syndrome of the model mice, attenuated colitis-activated NF-κB pathway measured by DNA-binding assay and colitis-elevated expression of proinflammatory cytokines examined with CCK cytotoxic kits, and upregulated CD4+CD25+Foxp3+ Treg in blood and mesenteric lymph nodes measured by flow cytometry. In conclusion, BL-hIL-10 as a novel oral hIL-10 delivery system has been successfully established and oral administration of BL-hIL-10 alleviated inflammatory damage of colonic tissue in the model mice by blocking the colitis-activated NF-κB pathway and upregulating CD4+CD25+Foxp3+ Treg in blood and mesenteric lymph nodes in mice.