The MAVS (Mitochondrial Antiviral Signaling) pathway is an important component of the innate immune system that plays a critical role in defending the host against viral infections. This pathway is primarily responsible for detecting the presence of viral RNA or other viral components and initiating an antiviral immune response. In teleost, Mavs -related studies are rare. In this study, we generated mavs knockout zebrafish (mavs(-/-)) with 11 bp deletion at the target site using CRISPR/Cas9-mediated technique. To elucidate the function of Mavs in viral infection, we injected viral hemorrhagic septicemia virus (VHSV) into wild- type (WT) and mavs(-/-) larvae (4 days post fertilization (dpf)) and adult fish , then we evaluated the mortality percentage and VHSV copy number. Our results exhibited that t he mavs gene began to be expressed from 2 cell stages and in situ hybridization revealed its unique expression site in the kidney, liver, and intestine. According to the qPCR results, the liver of adult zebrafish has the greatest mavs expression, followed by the intestine. The successful mavs mutant was created utilizing a CRISPR/Cas9-mediated technique, which resulted in a 67 bp shortened protein with an early stop codon. Furthermore, in 7dpf larvae downstream gene analysis, interferon regulatory factor 3 (irf3) , irf7, ifn-φ1 , tnf-α1 , and il-6 were found to be increased in mavs(-/-) compared to WT. The VHSV injection (300 TCID50/larva) caused death at 60 hours post-injection (hpi ) in both types of larvae, and the mortality percentage was higher in mavs(-/-) compared to WT throughout the experiment. Furthermore, at 24 and 48 hpi , the viral copy number was considerably higher in mavs(-/-) compared to WT. Further, t he mortality of mavs(-/-) and WT adult fish infected with 5 × 106 TCID50 /ml VHSV began at 6 dpi and 8 dpi and reached 100% and ~73% at the end of the experiment, respectively. The mean viral copy numbers from five individual zebrafish in each group at various time points revealed notable discrepancies in VHSV copy numbers between the groups on days beyond the first. Throughout this period, the VHSV copy number in WT fish remained significantly lower than that in mavs(-/-) mutants. In summary, our study, which utilized the deletion of mavs in zebrafish, provides compelling evidence that Mavs plays a critical role in immune regulation during viral infections.