κ-carrageenan (κ-CGN) is an important food additive that has been demonstrated to induce colitis in animal models. In the present study, the effects of κ-CGN were assessed using an in vitro co-culture system that contained intestinal epithelial Caco-2 cells and activated macrophage-like THP-1 cells. The results demonstrated that in single cultures of Caco-2 and THP-1 cells treated with κ-CGN, the cytotoxicity and the secretion levels of IL-1β, IL-6 and TNF-α were low. In the co-culture system, however, κ-CGN treatment resulted in apoptosis and reduced the transepithelial electrical resistance of the Caco-2 cell monolayers. The secretion levels of TNF-α, IL-1β and IL-6 from the two cell types were increased significantly by κ-CGN treatment. Furthermore, pretreatment of the co-culture system with anti-TNF receptor 1 antibody suppressed the κ-CGN-induced apoptosis and attenuated the changes in the levels of IL-6 and IL-1β in the Caco-2 monolayers. This study indicated that κ-CGN-induced TNF-α secretion is the main contributor to cellular damage in Caco-2 monolayers exposed to κ-CGN.