Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), in intestinal epithelial cells significantly contribute to inflammatory bowel disease (IBD) and colorectal cancer (CRC). Given our previous findings that TNF-alpha is upregulated in intestinal epithelial Caco-2 cells induced by skatole, a tryptophan-derived gut microbiota metabolite, the present study aimed to explore the relationship between skatole and IL-6, alongside TNF-alpha. Skatole elevated the promoter activity of IL-6 as well as TNF-alpha, and increased IL-6 mRNA expression and protein secretion. In addition to activating NF-kappaB, the NF-kappaB inhibitor BAY 11-7082 reduced skatole-induced cell survival and the mRNA expression of IL-6 and TNF-alpha. NF-kappaB activation was attenuated by the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 and the p38 inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. U126 and SB203580 also decreased the skatole-induced increase in IL-6 expression. When skatole-induced AhR activation was inhibited by CH223191, in addition to promoting NF-kappaB activation, IL-6 expression was enhanced in a manner similar to that previously reported for TNF-alpha. Taken together, these results suggest that skatole-elicited NF-kappaB activation induces IL-6 and TNF-alpha expression, although AhR activation partially suppresses this process. The ability of skatole to increase the expression of IL-6 and TNF-alpha may significantly affect the development and progression of these diseases. Moreover, the balance between NF-kappaB and AhR activation appears to govern the skatole-induced increases in IL-6 and TNF-alpha expression. Therefore, the present findings provide new insights into the mechanisms linking tryptophan-derived gut microbiota metabolites with colorectal disease.