RATIONALE: Protopine, an active alkaloid in Papaver somniferum L., was abundant in a well-known anticough traditional Chinese medicine preparation-Zhi-Ke-Bao tablets. Till now, the metabolism feature and anticough mechanism of protopine have not been fully elucidated, restricting its further development. METHODS: The metabolites of protopine in rats were profiled by using ultra-high performance liquid chromatography coupled with time-of-flight mass spectrometry, and its anticough targets and mechanism were predicted by network pharmacology. RESULTS: In rats, a total of 19 metabolites were identified following ingestion of protopine (21 mg/kg/day, i.g.), including 4 in plasma, 6 in urine, 5 in feces, 10 in liver, 2 in spleen, 4 in lung, 3 in kidney, 3 in heart, and 3 in brain. The main metabolic features were ring-opening, methylation, demethylation, glucuronidation, sulfation, and hydroxylation. Among them, methylation, sulfation, and hydroxylation of protopine in vivo were revealed for the first time. The network pharmacology results show that protopine and its metabolites regulate physiological activities by acting on STAT3, SRC, CASP3, MTOR, MMP9, ESR1, and other targets, involving PI3K-Akt signaling pathway, FoxO signaling pathway, and TNF signaling pathway, etc. CONCLUSIONS: The metabolic features of protopine and its potential mechanisms for anticough effects were outlined, providing data for further anticough pharmacological validation of protopine.