HCC (Hepatocellular Carcinoma) is a critical health issue worldwide. Our previous animal experiment has confirmed that blueberry malvidin-3-galactoside (M3G) can regulate the progression of HCC. In this study, feces samples from the same batch of mice were collected to explore the regulatory mechanism of M3G on intestinal microbiota and microbial TCA cycle metabolism KEGG pathway in HCC mice based on 16S rRNA sequencing and metagenomics. Our results showed that blueberryM3Gincreased the microbial diversity and regulated the structure of intestinal microbiota in mice, such as increasing the abundance of Clostridia (butyric acid-producing bacteria), Oscillospira and Ruminococcus, and reducing the abundance of pathogenic Erysipelotrichi. Compared with the group of liver cancer and 5-fluorouracil, blueberry M3G significantly regulated microbial TCA cycle KEGG pathway via improving the expression of key proteins (porA, DLAT, aceE, PC and OGDH). Additionally, we found which the abundance of Muribaculum intestinale increased by blueberry M3G may be an important factor affecting the microbial TCA cycle KEGG pathway via the pearson correlation (R) analysis of protein and microbiota. Taken together, these results demonstrate that the blueberry M3G has the potential to be an intestinal microbiota regulator and an adjuvant to HCC therapy.