Pyrrolizidine alkaloids (PAs) are the most common phytotoxins with documented human hepatotoxicity. PAs require metabolic activation by cytochromes P450 to generate toxic intermediates which bind to proteins and form protein adducts, thereby causing cytotoxicity. This study investigated the role of the gut-liver axis in PA intoxication and the underlying mechanisms. We exposed mice to retrorsine (RTS), a representative PA, and for the first time found RTS-induced intestinal epithelium damage and disruption to intestinal barrier function. Using mice with tissue-selective ablation of P450 activity, we found that hepatic P450s, but not intestinal P450s, were essential for PA bioactivation. Besides, in RTS-exposed, bile duct-cannulated rats, we found the liver-derived reactive PA metabolites were transported by bile into the intestine to exert enterotoxicity. The impact of gut-derived pathogenic factors in RTS-induced hepatotoxicity was further studied in mice with dextran sulfate sodium (DSS)-induced chronic colitis. DSS treatment increased the hepatic endotoxin level and depleted hepatic reduced glutathione, thereby suppressing the PA detoxification pathway. Compared to RTS-exposed normal mice, the colitic mice displayed more severe RTS-induced hepatic vasculature damage, fibrosis, and steatosis. Overall, our findings provide the first mode-of-action evidence of PA-induced enterotoxicity and highlight the importance of gut barrier function in PA-induced liver injury.