Extracellular vesicles (EVs) have garnered attention in research for their potential as biochemical transporters and immune modulators, crucial for regulating the host immune system. The present study was conducted to isolate and characterize EVs from Gram negative bacteria (EVs) and investigate their proteomic profile and immune responses. Isolation of EVs was carried out using ultracentrifugation method. Transmission electron microscopy results confirmed the spherical shape of EVs. The average size and zeta potential were 85.3 ± 1.8 nm and -8.28 ± 0.41 mV, respectively. EVs consisted of 1,487 distinct proteins. Subcellular localization analysis revealed that "cell" and "cell part" were the most predominant areas for protein localization. Proteins associated with virulence, along with several chaperones that facilitate protein folding and stability, were also present. No toxicity was detected when EVs were treated to fathead minnow (FHM) cells up to 100 μg/ml. Fluorescent-labeled EVs showed cellular internalization in FHM cells at 24 h post treatment (hpt). gene expression in Raw 264.7 cells showed upregulation of interleukin , , and interferon with simultaneous upregulation of anti-inflammatory . , gene expression revealed that except for heat shock protein , all other genes were upregulated suggesting that EVs induced the expression of immune-related genes. Western blot analysis showed increased protein levels of tumor necrosis factor (Tnf)α in EVs-treated spleen tissue of zebrafish. Our results confirm that EVs can be successfully isolated using the ultracentrifugation method. Furthermore, exploring immunomodulatory mechanism of EVs is essential for their potential use as novel therapeutics in fish medicine.