IFN production is crucial for hosts to defend against viral infection, yet it must be tightly controlled to maintain immune homeostasis. TANK-binding kinase 1 (TBK1) is a pivotal kinase in the IFN induction signaling pathway, but it is negatively regulated by multiple molecules to avoid the excessive expression of IFN in mammals. However, the identified TBK1 suppressors and the mechanisms are rare in fish. In this study, we show that zebrafish major vault protein (MVP) recruits and degrades TBK1 in a lysosome-dependent manner to inhibit IFN production. Through viral infection, polyinosinic:polycytidylic acid and RIG-I-like receptor factor stimulation upregulated IFN expression, but overexpression of MVP significantly subverted these inductions. On the protein level, MVP interacted with TBK1, and interestingly, MVP recruited TBK1 from a uniformly distributed state in the cytoplasm to an aggregated state. Finally, MVP mediated the lysosome-dependent degradation of TBK1 and decreased the IFN response and IFN-stimulated genes expression. Our findings reveal that zebrafish MVP is a negative regulator of IFN production by restricting the activation of TBK1, supplying evidence of the balanced mechanisms of IFN expression in lower vertebrates.