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Flotillin-2, an important protein of vesicular endocytosis, plays an essential role in a large number of cellular processes, including viruses and pathogen infection. In the present study, a flotillin-2 homolog in Litopenaeus vannamei, designed as Lvflotillin-2, was cloned and characterized. To analyze the putative role of Lvflotillin-2 during white spot syndrome virus (WSSV) infection, real-time quantitative PCR was performed. The result showed that the transcriptional level of Lvflotillin-2 was up-regulated significantly after virus challenge. Furthermore, upon WSSV stimulation, Lvflotillin-2 in shrimp cells could translocate from the plasma membrane to intracellular compartments, and unexpectedly, also into nucleus. Additionally, depletion of Lvflotillin-2 inhibited WSSV gene ie1 transcription. It suggested that Lvflotillin-2 could be hijacked by WSSV. These observations indicated that Lvflotillin-2 was involved in WSSV infection, and presented here should be useful for gaining insight into shrimp immunity and WSSV pathogenesis.Lvflotillin-2 transcription was significantly up-regulated upon WSSV challenge.Dynamic localization of Lvflotillin-2 was found in shrimp cells after WSSV challenge.Inhibition of Lvflotillin-2 decreased viral gene transcription.