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The clade B serpins contain predominantly intracellular proteins and were reported to be involved in multiple biological functions, especially in inflammation and immune system function. However, studies about the role of the invertebrate intracellular serpins in immune responses were still deficient. Therefore, this paper focused on the functional characterization of LvserpinB3 in white shrimp Litopenaeus vannamei. The pAc5.1–LvserpinB3-EGFP vector was transfected into Drosophila Schneider 2 (S2) cells to analyze the subcellular localization of LvserpinB3, and fluorescent imaging showed that LvserpinB3 were mainly localized to the mitochondria. Knockdown LvserpinB3 significantly increased the mRNA expression of LvSpätzle4 (LvSpz4) and LvPenaeidin4 (LvPen4) upon Vibrio anguillarum infection. Moreover, GST-Pull down analysis showed that LvserpinB3 could interact with serine protease 1 (LvSP1). The recombinant LvserpinB3 (rLvserpinB3) protein exhibited inhibitory roles on the proteolytic activity of trypsin, whereas, mutation at the P1 residue led to the disfunction of the inhibitor. Furthermore, the LvserpinB3 and trypsin mixture were incubated with Anti-SERPINB3 antibodies, and a peptide band with an apparent molecular weight of 30 kDa were detected by western blot analysis. These findings might be valuable in understanding the potential role for LvserpinB3 in inhibiting the target proteases during shrimp immune defences.LvserpinB3 was mainly localized to the mitochondria.LvserpinB3 might participate in the TLR-NF-κB pathway by blocking the activation of Spätzle.LvserpinB3 could bind to SP1 through targeting at the active sites.The reduction in trypsin activity probably results from being degraded as a normal substrate.