Background <p>Exosomes have emerged as promising vaccine candidates due to their role in intercellular communication and bioactive cargo-carrying capacity. However, their intrinsic properties in activating adaptive immunity have not yet been fully addressed. This study aims to elucidate how teleost peritoneal cell-derived exosomes (PDEs) elicit CD4⁺ T cell activation and contribute to vaccine-mediated adaptive immunity.</p> Methods <p>PDEs were isolated from flounder immunized with inactivated <i>Vibrio anguillarum</i> (Exo^FKC) or non-immunized flounder (Exo^Con) via differential centrifugation. Tissue and cellular distribution of DiO-labeled exosomes was visualized and analyzed via small animal imaging, fluorescence microscopy, and flow cytometry. The capacity of PDEs to promote CD4⁺ T cell activation was assessed using flow cytometry and CFSE assay. Multi-omics approaches were used to analyze Exo^FKC-mediated CD4⁺ T cell activation mechanisms at protein, mRNA, and miRNA levels. CD4⁺ T cell levels and antibody production were analyzed to assess the impact of Exo^FKC on enhancing vaccine potency.</p> Results <p>After intraperitoneal vaccination, PDEs increased markedly. These exosomes fused with peritoneal cells to facilitate the transport of antigen-presenting molecule locally and were transferred to key lymphoid organs. PDEs carrying T cell-associated RNA (<i>jun/p38a</i>) and miRNA (novel-m0034-3p), with elevated MHCII and CD80/86 levels, promoted CD4⁺ T cell activation. Exo^FKC, when used as an adjuvant with the vaccine, enhanced vaccine efficacy by activating CD4⁺ T cells and increasing antibody production.</p> Conclusions <p>This study highlights a novel mechanism by which exosomes directly contribute to vaccine-induced immunity, as demonstrated in a lower vertebrate model. It is plausible that similar processes may occur in higher vertebrates, including humans, providing new insights into the role of exosomes in adaptive immune responses.</p>