2,4-Dichlorophenol (2,4-DCP), an environmental pollutant, was reported to cause hepatotoxicity. The biochemical mechanisms of 2,4-DCP induced liver injury remain unknown. The present study showed that 2,4-DCP is chemically reactive and spontaneously reacts with GSH and bovine serum albumin to form GSH conjugates and BSA adducts. The observed conjugation/adduction apparently involved the addition of GSH and departure of chloride via the ipso substitution pathway. Two biliary GSH conjugates and one urinary N-acetyl cysteine conjugate were observed in rats given 2,4-DCP. The N-acetyl cysteine conjugate was chemically synthesized and characterized by mass spectrometry and NMR. As expected, 2,4-DCP was found to modify hepatic protein at cysteine residues in vivo by the same chemistry. The observed protein adduction reached its peak at 15 min and revealed dose dependency. The new findings allowed us to better understand the mechanisms of the toxic action of 2,4-DCP.