Rhomboid domain-containing protein 3 (Rhbdd3) has been identified as playing an essential role in innate mammalian immunity. However, the products and functions of the gene in aquatic animal cells, especially its antiviral characteristics, remain unknown. Spring viraemia of carp virus (SVCV) is a highly pathogenic agent, responsible for significant mortalities in several economically important Cyprinidae fish species. Infectious pancreatic necrosis virus (IPNV) is the causative agent of an acute, highly contagious, and destructive disease named infectious pancreatic necrosis (IPN). The cumulative mortality of young infected salmon can exceed 90%. SVCV and IPNV are difficult to eradicate and there are currently no effective therapeutic strategies or drugs for either viruses. Thus, new interventions need to be developed. Innate immunity also plays an important role in the protection of fish against early viral infections. In the present study, we aimed to obtain the coding region of the common carp ( gene. Furthermore, we sought to determine the function of Rhbdd3 in fish cells and explore the effects of Rhbdd3 on aquatic viral infections. Firstly, the gene of common carp was amplified by RT-PCR, with primers designed according to the conserved regions of the predicted fish gene was cloned into the eukaryotic expression vector pCI-neo, to construct the pCI- plasmid. Next, EPC (Epithelioma Papulosum Cyprinid) and CHSE-214 (Chinook Salmon Embryo) cells were transiently transfected with pCI-, and the expression of Rhbdd3 was detected using western blot analysis. The viability of the transfected cells was examined at 12 h, 36 h, and 72 h post transfection with a CCK-8 kit. SVCV and IPNV infections were conducted at 24 h post pCI- transfection. Viral productive replication was assessed via immunofluorescence microscopy, western blot, and RT-qPCR analyses. The results indicated that the ORF of common carp is 1050 bp, which encodes a putative peptide of 349 amino acids. Overexpression of Rhbdd3 was verified using western blot analysis post pCI- transfection, and the overexpression did not affect the viability of EPC and CHSE-214 cells. Meanwhile, the immunofluorescence microscopy, western blot, and RT-qPCR detection results all showed that Rhbdd3 overexpression inhibited SVCV and IPNV infections. Collectively, our data indicate that Rhbdd3 can inhibit fish viral infections without affecting fish cellular viability. The results presented in this study will facilitate the development of aquatic broad-spectrum antiviral drugs and provide a new insight into the function of Rhbdd3.