The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1β (IL-1β) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1β. We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1β intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1β following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1β were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed.