White spot syndrome virus (WSSV) is a large nuclear-replicating DNA virus of crustaceans such as shrimp and crayfish; however, the molecular mechanisms facilitating its transport from the invasion site to the cell nucleus have not yet been well elucidated. In this study, a CqProfilin (CqPFN) with a conserved PROF domain was identified from the red claw crayfish Cherax quadricarinatus. CqPFN was ubiquitously expressed in all examined tissues and hemocyte, with the highest levels in the hemocyte, followed by hematopoietic tissue (Hpt) from which the hemocyte were derived in crayfish. The transcript of WSSV genes such as IE1 and VP28 was obviously decreased both in vivo in hemocyte and Hpt, as well as in vitro in cultured Hpt cells, after CqPFN gene silencing; in contrast, the expression of viral genes was significantly increased by the introduction of a recombinant CqPFN protein in Hpt cells in vitro. Moreover, CqPFN was clearly colocalized with the main viral nucleocapsid protein VP664 and F-actin cytoskeleton, respectively, during the early stage of WSSV infection in Hpt cells. In addition, CqPFN was confirmed to interact with a truncated VP664^2,405-2,535 and another viral nucleocapsid protein VP15 of WSSV and Cqβ-Actin from Hpt by co-immunoprecipitation assays. Further studies found that VP664 also colocalized with F-actin in the Hpt cell cytoplasm after WSSV infection, suggesting that the actin cytoskeleton was involved in the intracellular transport of incoming viral nucleocapsid. Taken together, CqPFN might combine with the actin cytoskeleton to promote WSSV infection through binding with viral nucleocapsid proteins VP664 and VP15, promoting intracellular transport of viral incoming nucleocapsid for further releasing genome into the nucleus for transcription. Collectively, these results provided an understanding of the WSSV pathogenesis, which will contribute to the development of an antiviral strategy against WSSV disease.