Gut microbiota imbalance or gut dysbiosis emerges as a robust connection to hepatocellular carcinoma (HCC) through the gut-liver axis via enterohepatic circulation. However, current knowledge about gut microbiota in different HCC etiologies remains constrained. This study aimed to investigate the gut microbiota composition in patients with HCC at different etiologies. In this study, 17 patients with viral-related HCC, 13 HCC patients with non-viral-related HCC. and 10 healthy controls were recruited for comparative analysis. To examine gut microbiota profile, fecal samples were collected and 16S rRNA were sequenced using NGS. Then, data were analyzed by bioinformatic tools. Notably, a reduction of microbial diversity was observed in the HCC cohort relative to the healthy control group. Interestingly, there were 3 distinctive gut microbiomes including Bacteroides, Ruminococcus gnavus group and Erysipelatoclostridium, which showed significantly higher in the non-viral related HCC group when compared with viral-related HCC group. Based on PICRUSt2 analysis, these microbes were linked to the immune response such as lipopolysaccharide biosynthesis, thus contributing significantly to the progression of HCC. These results suggested that decreasing of gut microbiota diversity and increasing of those 3 bacteria could play an important role in HCC development. These bacteria could be used as diagnostic biomarkers in patients with HCC. In the future, interventions targeting the modulation of intestinal microbiota could enhance the intestinal equilibrium and might prevent the disease particularly in patients with HCC.