Backgrounds: Numerous algorithms based on patient genetic variants have been established with the aim of reducing the risk of GI bleeding and thromboembolism during warfarin administration. However, approximately 35% of individual warfarin sensitivity remains unexplained. Gut microbiota composition related to the vitamin K generation should be taken into account. Methods : Faecal samples were collected from 200 inpatients undergoing heart valve replacement (HVR). Ultimately, faecal samples from 80 inpatients (27 low responders (LRs), 27 high responders (HRs) and 26 normal responders (NRs) were analyzed for microbiota composition through 16S rDNA sequencing. Fifteen samples (5 LRs, 5 HRs and 5 NRs) were also analyzed through metagenomic whole-genome shotgun (WGS) sequencing. We validated the results from 10 LRs, 10 HRs and 10 NRs. Results: Significant differences were observed in the diversity and composition of the gut microbiome among the three groups. The genera Bacteroides, Escherichia-Shigella and Klebsiella were enriched in the LRs, while the genus Enterococcus was enriched in the HRs. WGS sequencing indicated that the abundance of enzymes, modules and KO associated with bacterial vitamin K biosynthesis was significantly higher in LRs than in HRs or NRs. The 12 optimal microbial markers were identified through tenfold cross-validation with a random forest model. Conclusions: This study characterized gut microbiome in the different response to warfarin of HVR patients and suggested that gut microbiome might play an important role in the clinical warfarin anticoagulation therapy.