A role of the mitochondrial dynamin-related protein (Drp1) on gut microbiome composition and intestinal barrier function after hemorrhagic shock has not been identified previously and thus addressed in this study. Here, we used a combination of 16S rRNA gene sequencing and mass spectrometry-based metabolomics profiling in WT and Drp1 KO mouse models to examine the functional impact of activated Drp1 on the gut microbiome as well as mitochondrial metabolic regulation after hemorrhagic shock. Our data showed that changes in mitochondrial Drp1 activity participated in the regulation of intestinal barrier function after hemorrhagic shock. Activated Drp1 significantly perturbed gut microbiome composition in the Bacteroidetes phylum. The abundance of short-chain fatty acid (SCFA) producing microbes, such as Bacteroides, Butyricimonas and Odoribacter, was markedly decreased in mice after shock, and was inversely correlated with both the distribution of the tight junction protein ZO1 and intestinal permeability. Together, these data suggest that Drp1 activation perturbs the gut microbiome community and SCFA production in a ROS-specific manner and thereby substantially disturbs tight junctions and intestinal barrier function after hemorrhagic shock. Our findings provide novel insights for targeting Drp1-mediated mitochondrial function as well as the microbiome in the treatment of intestinal barrier dysfunction after shock.