Lumpfish (Cyclopterus lumpus) is a native fish of the North Atlantic Ocean used as sea lice biocontrol in Atlantic salmon farms. Lumpfish also has been used as model for marine infectious diseases and immunity. Lumpfish is susceptible to Vibrio anguillarum infection, and upon infection, lumpfish immunity is activated to preclude the disease progression. Extracellular vesicles (EVs) play an important role in early immune cellular communication. Lumpfish EVs and their potential role in immunity have not been explored. Herein, EVs where isolated from serum of naïve lumpfish and from lumpfish infected with V. anguillarum at 5 and 10 days post infection (dpi). EVs characteristics were studied by electron microscopy and nanoparticle tracking, and protein cargo was analysed by Western blot and proteomic analysis. The isolated EVs showed a spherical shape ranging from ∼30 nm to 300 nm in diameter, but at 5 dpi the size variation was higher. A total of 395 proteins were identified. Upregulated proteins were linked to complement pathway/innate immunity, heme/iron binding, defense response to bacterium, apoptotic signaling pathway, and actin binding. Downregulated proteins were associated with ribonucleoprotein/ribosomal protein, transport and translation elongation factor activity, acute phase, protein phosphorylation and apoptotic process. Upon infection V. anguillarum infection, lumpfish EVs cargo was modified, from transporting metabolic proteins to proteins related to immunity. Characterization of peripheral lumpfish EVs protein profile during V. anguillarum infection provided with potential biomarkers repertoire that could be utilised in the development of novel tools to diagnose and control of V. anguillarum infection in finfish aquaculture.