Abstract3,3′,4,4′,5‐pentachlorobiphenyl (PCB126) is widely distributed, non‐degradable and bioaccumulative, which can affect the function of tissues and organs of the living organisms. Melatonin (MT) is a sort of indole neurohormone that is mainly secreted by the pineal gland. Numerous studies have shown that MT can alleviate intestinal injury through various mechanisms such as antioxidant, anti‐inflammatory, and anti‐apoptosis. For the above reasons, the aim of this study is to explore the mechanism of intestinal injury in mice after exposure to PCB126 as well as the antagonistic effect of MT. Mice were respectively fed PCB126 (0.326 mg/kg) and/or MT (10 mg/kg) in vivo. In vitro, colonic epithelial cells (MCEC) were treated with PCB126 (150 μM) and/or MT (2 mM). We found that the microscopic structure of colon tissue was impaired after exposure to PCB126. The levels of oxidative stress, the protein and mRNA levels of expression of inflammatory related factors were significantly increased and the expression levels of intestinal tight junction protein were decreased. Notably, MT can promote Nrf2/HO‐1 expression level and reduce the colonic injury caused by PCB126. Further in vitro treatment with reactive oxygen species inhibitors (NAC) showed that it significantly alleviated PCB126‐induced in MCEC cell damage. In summary, the above results suggested that MT alleviates PCB126‐induced colon inflammation by inhibiting the overproduction of reactive oxygen species (ROS) and up‐regulating the expression level of intestinal tight junction protein. Our results contribute to the further comprehension of the intestinal toxicity effects of PCB126 and the significant role of MT in preserving the mechanisms of intestinal injury.