Pacsin1 is a crucial protein involved in vesicle formation and transport, and its role in neuronal development and cytosolic dynamics has been extensively studied. However, its involvement in immune regulation still needs to be better understood. In this study, we show that pacsin1 exerts a negative regulatory effect on RLR-mediated signaling pathways activated by SCRV or poly(I:C), thereby inhibiting MITA-mediated antiviral responses. Mechanistically, pacsin1 facilitates the degradation of MITA, thus impeding immune signaling. Additionally, overexpression of pacsin1 promotes the conversion of LC3B-I to LC3B-II, while treatment with the autophagy inhibitor ammonium chloride results in the accumulation of LC3B-II and prevents pacsin1-mediated MITA degradation. Our findings suggest that pacsin1 targets MITA for autophagic degradation, thereby suppressing the innate antiviral response in fish.