<p>This study investigated the impact of antibiotics on the therapeutic efficacy of PD-1 inhibitors and their association with the intestinal microbiota. Using a Hepa1-6 hepatocellular carcinoma mouse model, various antibiotics (vancomycin, colistin, and combination antibiotics) were administered alongside a PD-1 inhibitor. The results indicated that antibiotic treatment significantly altered the intestinal microbiota composition, with vancomycin and the combination antibiotics notably reducing PD-1 inhibitor efficacy. 16S rRNA sequencing revealed that antibiotic-induced selective pressure profoundly impacted both the diversity and abundance of the microbiota. Specifically, the relative abundance of <i>Bacteroidetes</i> and <i>Firmicutes</i> was significantly diminished following vancomycin treatment, correlating with reduced PD-1 inhibitor efficacy. Further analysis of the tumor immune microenvironment revealed no significant changes in the CD8⁺ T cell ratio, TGF-β, IL-6, IL-17, or PD-1 levels. However, the proportion of CD4⁺ T cells was markedly lower in the combination antibiotic group, and the expression of pro-inflammatory cytokines IFN-γ and IL-2 was substantially decreased across all antibiotic-treated groups. These findings suggest that alterations in specific intestinal bacterial populations, likely through modulation of pro-inflammatory cytokine levels in the tumor immune microenvironment, compromise immunotherapy effectiveness. This study highlights the critical role of healthy intestinal microbiota in optimizing tumor immunotherapy efficacy and emphasizes the need for careful consideration of antibiotic use in such treatments. Moreover, it provides novel insights into the mechanisms by which antibiotics may interfere with immunotherapy, laying the groundwork for optimizing anti-tumor immunotherapy strategies.</p> Graphical Abstract <p></p>