Here, with an aim to determine the relationship between gut microbiota and HCC development in both sexes, and to screen potential microbial biomarkers for HCC diagnosis, we studied the changing patterns in the gut microbiota from mice of both sexes with liver-specific knockout of Tsc1 (LTsc1KO) that spontaneously developed HCC by 9–10 months of age and compared them to the patterns observed in their wide-type Tsc1fl/fl cohorts using high-throughput sequencing. Here, TSC1 is linked to hepatocellular carcinoma.