Abstract: Spring viraemia of carp virus (SVCV) is an OIE listed highly pathogenic agent of spring viraemia of carp (SVC), which can cause a high morbidity and mortality in several economically important Cyprinidae fish species. SVC outbreaks occur mostly in the spring with water temperature below 17℃. The mortality of infected young carp can reach up to 90%. Currently, there are no licensed vaccines or therapeutics for SVCV. Thus, elucidation of the SVCV induced host factors against viral infection and its anti-viral mechanisms would be helpful for the development of effective interventions. We previously found that programmed cell death 6-interacting protein (Pdcd6ip) was significantly upregulated in the SVCV-infected EPC cells based on proteomic analyses. Pdcd6ip, also known as Alix or Aip1, has been identified to function in a number of pathways. It is a central component of the endosomal sorting complexes, which is responsible for sorting membrane proteins into vesicles that bud into the multivesicular bodies. Meanwhile, it is involved in apoptosis through caspase 9 activation following endoplasmic reticulum stress. Reports also indicate Pdcd6ip play a key role in the regulation of viral replication in many viruses, such as HIV, EIAV and VSV. To determine the role of Pdcd6ip in SVCV replication, the common carp (Cyprinus carpio) pdcd6ip gene was amplified by reverse transcript-PCR, and the coding region of pdcd6ip was cloned into the pRSET-A prokaryotic expression vector. Recombinant protein was induced, purified, and used to immunize rabbits. The specificity of Pdcd6ip antibody was verified by western blot. Next, the plasmid pCI-pdcd6ip for eukaryotic expression of Pdcd6ip, was constructed and transiently transfected into EPC cells. The expression of Pdcd6ip was verified by RT-qPCR and western blot. The viabilities of the transfected cells were examined at 24h, 48h and 72h post transfection with a CCK-8 kit. The results showed that overexpression of Pdcd6ip did not affect the proliferation of EPC cells. Then, the Pdcd6ip overexpressed cells were infected with SVCV at 24h post transfection, and the replication of this virus was detected by RT-qPCR, immunofluoresence microscopy, western blot and plaque assay. All results indicated that overexpression of Pdcd6ip significantly inhibited SVCV infection. The results provide a new insight into the development of anti-SVCV drugs.