While FOLFOX has been the preferred chemotherapeutic strategy in colon cancer treatment, the limited response rate has seriously restricted its application in clinic. The underlying mechanisms of the individual response of FOLFOX remain to be elucidated. In this study, pharmacomicrobiomics integrated with pharmacometabolomics was applied to disentangle the key role of specific gut bacteria and microbiota derived metabolites involved. As a result , significant variation of chemotherapy efficacy was observed in tumor bearing mice after FOLFOX administration. 16S rRNA gene sequencing analysis revealed the relative abundance of Staphylococcus , Jeotgalicoccus , Sphingomonas significantly increased in the FOLFOX sensitive group, whereas Prevotella was higher in the non sensitive individuals . Meanwhile, verification study o n FOLFOX combined with bacteria colonization indicates that Prevotella could attenuate the anti cancer efficacy of FOLFOX in vivo . Furthermore, gut derived metabolite 3-Oxocholic acid (3-Oxo) identified by metabolomics approach was confirmed to associate with Prevotella in fecal samples. In addition, preliminary functional exploration suggested that 3-Oxo could reverse the anti cancer effect of FOLFOX and promote malignant progression Taken together, integrated pharmacomicrobiomics and pharmacometabolomics revealed that Prevotella and related metabolite 3-Oxo may be responsible for individualized FOLFOX efficacy, which provides novel predictive biomarkers for FOLFOX precise medicine as well as targets for colon cancer therapy.