Objective To isolate and identify the main components of extracellular vesicles (EV) secreted by Trichinella spiralis newborn larvae and elucidate the function of these EVs. Methods T. spiralis muscle larvae were sourced from the muscles of mice, which had been utilized for T. spiralis preservation. The small intestine was excised 5 days after infected with muscle larvae, and T. spiralis adults were obtained through mesh screening. These adults were then cultured in RPMI 1640 medium for 48 hours to yield newborn larvae. EVs were collected by differential ultracentrifugation from newborn larvae culture fluid of T. spiralis, and were observed morphologically under transmission electron microscope, and the diameter of the newborn larvae EVs was measured by nanoparticle tracking analysis. The EV protein components were verified using Western blotting, and analyzed by proteomic mass spectrometry. RNA sequencing technology was applied to analyze the miRNA sequences of the newborn larvae and their secreted EVs. The differences of protein components and differential expression between newborn larvae and their EVs were compared using t-test. The differentially expressed proteins and miRNA target genes were analyzed by gene ontology (GO) functional item enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. WoLFPSORT was used to analyze the subcellular localization of proteins. Results Transmission electron microscopy and nanoparticle tracking analysis revealed that T. spiralis newborn larvae produce cup-shaped, membranous vesicles with a diameter of approximately (101.7 ± 1.8) nm. Western blotting demonstrated that the newborn larvae-derived EVs generate specific bands when reacted with sera from mice infected with Trichinella spiralis, with a approximate molecular weight of 65 000. Through proteomics mass spectrometry, 1 424 and 231 proteins were detected in the newborn larvae and EV samples, respectively. Among these, 220 proteins were expressed in both groups, with 116 displaying differential expression patterns (31 upregulated and 85 downregulated in EVs). The subcellular localization analysis revealed that the differentially expressed proteins were predominantly localized in the cytoplasm (30.2%, 35/116), nucleus (19.0%, 22/116), and cell membrane (17.2%, 20/116). miRNA sequencing data showed that 183 and 117 miRNAs were detected in newborn larvae and EV samples, respectively; 46 miRNAs were found in both groups, with 40 showing differential expression patterns (10 upregulated and 30 downregulated in EVs). Notably, let-7-5p, miR-125-5p, miR-10, and miR-92 exhibited higher levels of upregulation in newborn larvae-derived EVs. The GO functional enrichment analysis indicated that the differentially expressed proteins could be categorized into three main groups: cell composition (57), molecular function (73), and biological processes (63). The target genes of differentially expressed miRNA were primarily associated with autophagy-related genes such as transferase activation of autophagy-related 12 and ubiquitin linkage enzyme complex. KEGG pathway enrichment analysis suggested that the differentially expressed proteins were significantly enriched in metabolic pathways and protein processing in endoplasmic reticulum-related pathways, whereas the target genes of differentially expressed miRNA were only involved in the autophagy-other pathway. Conclusion This study successfully isolated and identified the EVs secreted by newborn larvae of T. spiralis. 116 differentially expressed proteins and 40 miRNAs were identified between the newborn larvae and their EVs, predominantly participating in metabolic, autophagic and other pathways, which inferring that newborn larvae EVs may play important roles in evading host immune defence at early Trichinella infection.