The aims of this study were to analyze the structure and diversity of intestinal microorganisms in wild Anguilla japonica eel larvae from the Yangtze River Estuary and to explore the function of these intestinal microorganisms. MiSeq 16S rRNA high-throughput sequencing technology was used to sequence the 16S rRNA of microorganisms in wild eel larvae at different pigment development stages from January to April. We then counted the number of operational taxonomic units (OTU) of intestinal microorganisms in the samples, analyzed species composition, abundance, and alpha diversity, and predicted the function of these intestinal microorganisms. A total of 1467 OTUs were identified from the Japanese eel larvae samples, with an average of 396 per sample; these microorganisms were determined to belong to 51 phyla, 140 classes, 286 orders, 414 families, 643 genera, and 959 species. Each sample had an average of 26 phyla and 229 genera; however, a significant decrease in this diversity was observed with increasing months. At the phylum level, the dominant microflora were Proteobacteria (81.33%) and Bacteroidota (10.61%). At the genus level, the dominant bacterial groups were Psychrobacter (35.10%), Pseudomonas (16.75%), Flavobacterium (7.99%), Achromobacter (4.82%), and Shewanella (4.21%), which differed from the intestinal microbial communities observed in other fishes. The abundance and diversity of intestinal microflora in eel larvae from the January group were significantly higher than those in other months (P＜0.05). Nonetheless, there was no significant difference in the structure and diversity of the intestinal microbial community among the other three groups or between the VA and VB stages of eel larvae at different pigment development stages (P＞0.05). According to KEGG path annotation, it was predicted that most of the genes encoded by the intestinal microflora of Japanese eel larvae were related to metabolism; further, the relative abundance of amino acid transport and metabolic functional groups was high. The composition of microorganisms in the intestinal tract of Japanese eel larvae is complex; therefore, the analysis of these intestinal microorganisms and their corresponding functions helps develop our understanding of the influence of intestinal microflora on Anguilla japonica feeding, thereby providing a theoretical basis for further studies on fish diet.