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In shrimp, several glutathione peroxidase (GPX) genes have been cloned and functionally studied. Increasing evidence suggests the genes’ involvement in white spot syndrome virus (WSSV)- or <i>Vibrio alginolyticus</i>-infection resistance. In the present study, a novel GXP gene (<i>LvGPX3</i>) was cloned in <i>Litopenaeus vannamei</i>. Promoter of <i>LvGPX3</i> was activated by NF-E2-related factor 2. Further study showed that <i>LvGPX3</i> expression was evidently accelerated by oxidative stress or WSSV or <i>V. alginolyticus</i> infection. Consistently, downregulated expression of <i>LvGPX3</i> increased the cumulative mortality of WSSV- or <i>V. alginolyticus</i>-infected shrimp. Similar results occurred in shrimp suffering from oxidative stress. Moreover, LvGPX3 was important for enhancing <i>Antimicrobial peptide</i> (<i>AMP</i>) gene expression in S2 cells with lipopolysaccharide treatment. Further, knockdown of <i>LvGPX3</i> expression significantly suppressed expression of AMPs, such as <i>Penaeidins 2a</i>, <i>Penaeidins 3a</i> and <i>anti-lipopolysaccharide factor 1</i> in shrimp. AMPs have been proven to be engaged in shrimp WSSV- or <i>V. alginolyticus</i>-infection resistance; it was inferred that LvGPX3 might enhance shrimp immune response under immune challenges, such as increasing expression of AMPs. The regulation mechanism remains to be further studied.