Warfarin is a commonly prescribed anticoagulant in clinic. It has large individual variability. The pharmacokinetics and pharmacodynamics (PKPD) of warfarin had been established. There also exist some unknown factors that could affect warfarin effect. The purpose of the study was to explore the contribution of vitamin K and gut microbiota to individual variability of warfarin in cardiac surgery patients. A total of 246 patients were enrolled in the present study. Serum and fecal samples were collected used to detect warfarin and vitamin K (VK1 and MK4) concentrations and the diversity of gut microbiota, respectively. The demographic characteristics, drug history, and CYP2C9 and VKORC1 genotype were recorded from the patients’ medical records. The INR and warfarin concentration bias were predicted according to the PKPD model. The results of INR and warfarin concentration predicted bias by the PKPD model were: 78.7% of Ideal prediction for INR, 66.7% of Ideal prediction for S-warfarin, 75.0% of Ideal prediction for R-warfarin. Only INR PE was compared with VK concentration and gut microbiota due to INR was the main indicator for warfarin therapy in clinic. The INR of patients decreased with increasing of VK concentration. The pharmacodynamic parameter C50 of S-warfarin, which was derived from the PKPD model, increased with increasing of VK concentration. The results diversity of gut microbiota showed that Prevotella might be associated with warfarin anticoagulation. In conclusion, vitamin K had some effect on the individual variability of warfarin. The gut microbiota might also have a certain effect on the variability of warfarin.