Background and Purpose Farfarae Flos (FF), derived from the flower buds of Tussilago farfarae L., is used as a traditional folk medicine for the treatment of cough, bronchitis and asthmatic disorders. However, its antitussive mechanism is unknown yet. Experimental Approach The antitussive mechanism of FF was investigated from the perspective of gut microbiota. The antibiotic pretreatment and fecal microbiota transplantation (FMT) were used to investigated the microbiota-host interaction after FF treatment. The composition of gut microbiota was detected by using 16S ribosomal RNA (rRNA) sequences. The gut metabolites variations were evaluated by LC-MS based metabolomic analysis and GC-MS based targeted short-chain fatty acids (SCFAs) profiling. Key Results FF increased the abundance of SCFAs-producing bacteria, which consequently led to the elevated SCFAs levels. SCFAs supplementation further confirmed the importance of SCFAs for the antitussive effect of FF. The antitussive effect of FF was gut microbiota dependent, as demonstrated by antibiotic treatment and FMT. FF could also probably regulate Argnine/NO metabolism by inhibiting arginase which was confirmed by antitussive effect of Argnine supplementation. Caffeic acid (CA) and quinic acid (QA), intestinal metabolites of caffeylquinic acids metabolized by Lactobacillus and Bifidobacterium, showed antitussive effect by increasing the butyrate, and butyrate-producing bacteria (Clostridium and Ruminococcus) were also enriched by QA. Conclusion and Implications Our study shows that CA and QA were proved as the key bioactive metabolites for the antitussive effect of FF. This study may present a novel approach for investigating the antitussive mechanism of herbal drug from the perspective of gut microbiota.