Tri-spine horseshoe crabs (HSCs)Tachypleus tridentatushave been facing the threat of population depletion for decades, and the physiology and microbiology of their early life stages are lacking. To explore what directs the change of juvenileT. tridentatusgut microbiota and how gut microbiota change, by using 16S rRNA sequencing of gut samples we detected the intestinal microbiome of juvenile HSCs and compared the impact of initial molting and initial feeding, as well as the effect of environment. Results showed that the predominant phyla in the gut microbial community of juvenile HSCs areProteobacteriaandBacteroidetes. The richness and diversity of intestinal microbes greatly decreased after initial molting. Microbial-mediated functions predicted by PICRUSt showed that "Signal Transduction", "Cellular Processes and Signaling", "Infective Diseases" and "Digestive System" pathways significantly increased in 2nd instars. As for the effect of environment, the connection between living environment and the intestinal microbiome started to manifest after initial molting. Unexpectedly, initial feeding treatment slightly affected the intestinal microbiome ofT. tridentatusin the early life stage, whereas the effect of initial molting was significant. The present study provided the first insight into the gut microbiota ofT. tridentatus, and the findings led a new sight to explain what guide the change of gut microbiota.