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Introduction: To establish a new model for exploring the mechanism of the gut microbiome and drug metabolism, we explored whether Taohong Siwu Decoction acts after metabolism by intestinal flora under the premise of clarifying the interaction between intestinal flora and drug metabolism. Methods: Taohong Siwu Decoction (TSD) was fed to germ-free mice and conventional mice, respectively. The serum from both groups of mice was removed and co-cultured with glioma cells in vitro. The co-cultured glioma cells were compared separately for changes at the RNA level using RNA-seq technology. The genes of interest in the comparison results were selected for validation. Results: The differences in the phenotypic alterations of glioma cells between serum from TSD-fed germ-free mice and normal mice were statistically significant. In vitro experiments showed that Taohong Siwu Decoction-fed normal mouse serum-stimulated glioma cells, which inhibited proliferation and increased autophagy. RNA-seq analysis showed that TSD-fed normal mouse serum could regulate CDC6 pathway activity in glioma cells. The therapeutic effect of TSD is significantly influenced by intestinal flora. Conclusion: The treatment of tumors by TSD may be modulated by intestinal flora. We established a new method to quantify the relationship between intestinal flora and the regulation of TSD efficacy through this study. [ABSTRACT FROM AUTHOR]