We examined the in vitro and in vivo effects of a probiotic, Escherichia coli strain M-17 (EC-M17), on NF-κB signalling, cytokine secretion and efficacy in dextran sulfate sodium (DSS)-induced murine colitis. NF-κB signalling was assessed using an NF-κB luciferase reporter cell line that was stimulated with TNF-α (100 ng/ml). p65 Nuclear binding and cytokine secretion (TNF-α, IL-1β and IL-6) were evaluated using a RAW 264·7 macrophage cell line that was exposed to lipopolysaccharide (LPS; 5 μg/ml). Mice were administered vehicle, EC-M17, metronidazole, or EC-M17 plus metronidazole for 13 d. During the final 6 d, mice also received 2 % DSS. Parameters evaluated included disease activity index (DAI), histology, myeloperoxidase and NF-κB p65. EC-M17 dose dependently inhibited TNF-α-induced NF-κB signalling. At 5 × 109 colony-forming units/ml, EC-M17 inhibited NF-κB by >95 %. LPS-induced nuclear p65 binding was significantly inhibited (78 %; P < 0·05) in RAW 264·7 macrophages at 1 × 108 colony-forming units/ml. EC-M17 also inhibited (by >90 %) the LPS-induced secretion of TNF-α, IL-1β and IL-6. In mice with DSS-induced colitis, EC-M17, metronidazole, and EC-M17 plus metronidazole significantly reduced DAI and colonic histology scores. Both EC-M17 and metronidazole reduced colonic IL-12, IL-6, IL-1β and interferon-γ. The combination of EC-M17 plus metronidazole resulted in more substantial cytokine reductions than were found with either treatment alone, and combination therapy significantly (P < 0·05 in both cases) reduced IL-1β compared with EC-M17 and colonic histology scores compared with metronidazole. Alone, and in combination with metronidazole, EC-M17 improved murine colitis, probably due to an inhibitory effect on NF-κB signalling.