Chronic intestinal inflammation contributes to an increased risk of colon cancer. Lactosucrose (LS), a kind of functional trisaccharide, can modulate immunity and promote microbe growth. The aim of this study was to investigate the effect of LS on 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colitis in rats. Rats were randomly divided into four treatment groups: the normal group, TNBS group, LS group, and salicylazosulfapyridine (SASP) group for five weeks. LS supplementation ameliorated TNBS-induced colitis. LS supplementation increased IL-10 production and suppressed the secretion of IL-12 in the colon, as compared to the TNBS group. LS decreased the production of TLR-2 protein and nuclear NF-κB p65 protein, as well as mRNA levels, as compared with colitic rats. These results indicate that chronic feeding of LS inhibited TNBS-induced chronic inflammation. LS has potential nutraceutical intervention to combat colitis.