Abstract In this study, GST Pull-down and mass spectrometry was applied to the precipitation and identification of the small GTP-binding protein (Rab7) interacting protein in hemocyte of Litopenaeus vannamei. According to the search in GenBank with the peptide mass fingerprint, the 45 kDa protein which was pulled down with the GST-tagged Rab7 (GST-Rab7, GTP bound form) was identified to be β-actin with 28% coverage of amino acid sequences. The interaction of Rab7 with β-actin was verified by both GST Pull-down and ELISA in vitro. Meanwhile, confocal microscopic observation showed that Rab7 could be co-localized with β-actin in hemocytes at 12 h post white spot syndrome virus (WSSV) infection (hpi). GST Pull-down and western blotting were used to analyze the cross-interaction between WSSV VP28, Rab7 and β-actin. The results showed that the GST-VP28, His-tagged Rab7 (His-Rab7) and His-β-actin formed a tripartite complex. At 12 hpi, confocal microscopic observation showed that WSSV could be co-localized with Rab7 and β-actin in hemocytes respectively. Furthermore, based on the in vivo neutralization assay, recombinant His-β-actin accelerated the infection of WSSV, conversely, recombinant His-Rab7 delayed WSSV infection in shrimp. These results suggested the interaction of Rab7 with β-actin and this interaction was involved in WSSV infection. Highlights • GST pull-down was used to precipitate the Rab7 interacting proteins in hemocyte. • The precipitated 45 kDa protein was identified to be Litopenaeus vannamei β-actin. • GST pull-down and ELISA confirmed the Rab7 binding to β-actin. • GST-VP28, His-Rab7 and His-β-actin form a tripartite complex in vitro. • Both Rab7 and β-actin was involved in WSSV infection. [ABSTRACT FROM AUTHOR]