The serine/threonine protein kinase MST4 plays multiple roles in the regulation of signaling pathways that govern cellular processes including mitosis, migration, homeostasis, polarity, proliferation, differentiation and apoptosis. Here we report the identification and characterization of the full-length sequence of LvMST4 from the shrimp L. vannamei, and investigations into its role in the shrimp's immune response to infection by the pathogenic bacterium Vibrio alginolyticus. Subcellular localization assays demonstrated the enzyme's presence in the shrimp's cytoplasm, and tissue-specific expression analysis revealed that it is expressed ubiquitously but at different levels in different tissues. Infection with V. alginolyticus increased LvMST4 expression and induced a rapid response via the TLR-TRAF6 signaling pathway, causing a decline in the total hemocyte count (THC) and an increase in respiratory burst (RB) activity. In non-infected shrimp, RNAi silencing of LvMST4 with dsRNA had no significant effect on THC but seemed to activate the TRAF6-MKK6-p38 pathway and reduced RB activity. In shrimp challenged with V. alginolyticus, LvMST4 silencing reduced bacterial clearance and increased the initial upregulation of LvTRAF6 while reducing the expression of LvMKK6 and Lvp38. LvMST4 silencing also slightly reduced the THC but caused pronounced increases in RB activity and cumulative mortality. These findings suggest that LvMST4 contributes to antimicrobial responses via the TLR-TRAF6 signal pathway, and helps maintain immunological homeostasis in L. vannamei.