Until now, there has been little research on the intestinal microbial community of horseshoe crabs. To fill this gap, we investigated the microbiome composition of the Chinese horseshoe crab,Tachypleus tridentatus, and the mangrove horseshoe crab,Carcinoscorpius rotundicauda. We sequenced the 16S rRNA gene of intestinal bacterial species and compared the microbial community structure and diversity. Next, we show that the total effective bacterial sequence was 36,865 reads, and the average annotated operational taxonomic unit (OTU) number was 240. Through hierarchical clustering analysis and principal coordinate analysis samples from two horseshoe crab species, we found that the intestinal flora of the same horseshoe crab species was relatively concentrated, while the microbiome of a different horseshoe crab species were significantly separated. Cluster analysis showed that two samples, one from Chinese horseshoe crabs and one from mangrove horseshoe crabs, had similar microbial community structure, while other samples were relatively discrete. The gut microbiota of the mangrove horseshoe crab were dominated by the phyla Tenericutes (42.71%), Firmicutes (24.27%), and Proteobacteria (20.39%), while the top three phyla in the Chinese horseshoe crab intestinal tract were Tenericutes (57.19%), Proteobacteria (22.14%), and Bacteroidetes (7.38%). To intuitively understand the similarity and overlap of the OTU composition of each group, we performed Venn diagram analysis. The two species shared 284 OTUs, accounting for 81.8% of the total. This indicates that although there is high similarity between mangrove and Chinese horseshoe crab in gastrointestinal microbial community structure, there are also some differences, which deserve further discussion.