Objective:To identify the expression level of known microRNA (miRNA) by high-throughput sequencing and analysis of the miRNA of Schistosoma japonicum schistosomula, and predict the miRNA target genes and their biological functions. Methods:Schistosoma japonicum schistosomula were prepared in vitro, and total RNA of schistosomula were extracted and analyzed to construct a library for performing high-throughput sequencing. The difference of miRNA expression was analyzed by using DEGseq R language package and perl script. Then the target genes and their biological functions of differential miRNA were predicted by miRanda software, Blast software, and KEGG database respectively. Results:There were 38 483 matching sequences in the miRNA expressed of Schistosoma japonicum schistomula in the constructed library compared with the latest miRBase database, and 60 miRNA were identified, sja-miR-125b was the miRNA with the highest expression, followed by sja-miR-61, sja-miR-71a, sja-miR-36-3p and sja-miR-10-5p, which accounted for 91% (3 263/3 585) of the total miRNA expression. MiRanda software predicted a total of 7 176 target genes, gene functions concentrated on nucleotide transferase activity, cellular nitrogen complex metabolism, molecular function, biological processes, biosynthesis, plasma membrane and protein maturation. The functional enrichment analysis showed that the highly expressed miRNA were mainly involved in pathogenic process, biological progress and multiple metabolic regulation pathways. Conclusions:The miRNA expressed significantly of Schistosoma japonicum schistomula mainly involved in the regulation of metabolic pathways during the differentiation, growth and pathogenesis of Schistosoma japonicum. To lay a foundation for the study of the regulatory mechanism of Schistosoma japonicum development and the development of new drugs.