In summary, on the basis of l-isoleucine treatment effectively ameliorating the TNF-α-induced inhibition of cell viability in IEC-18 cells, l-isoleucine administration assuaged the negative effect of drinking the DSS-supplementing water on growth performance and colonic health, and reduced, to some extent, the DSS-induced colitis in rats, which could be on account of l-isoleucine treatment relieving the inflammation via regulating the TLR4/MyD88/NF-κB pathway. The gene discussed is TLR4; the disease is colitis.