The thioredoxin system plays essential roles in maintenance and regulation of the redox state of cysteine residues in cellular proteins. The thioredoxin-related protein of 14 kDa (TRP14) is an important member of the TRX superfamily which acts on various substrate proteins, some of which are not overlapped with those of thioredoxin. The knowledge on the function of TRP14 in invertebrates is limited to date. In this study, a TRP14 gene was identified from Pacific white shrimp Litopenaeus vannamei (LvTRP14) and its role in immune responses was investigated. We demonstrated that the expression level of LvTRP14 was high in hepatopancreas and intestine, low in eyestalk, and medium in other tissues of healthy shrimp. The transcription of LvTRP14 in vivo was significantly down-regulated in Relish-silencing shrimp but up-regulated in STAT-silencing shrimp, indicating a complex regulation of LvTRP14 expression. Although the LvTRP14 expression showed little change after immune stimulation with different type of pathogens, knockdown of LvTRP14 expression using RNAi strategy could significantly facilitate the infection of white spot syndrome virus (WSSV) and Vibrio parahaemolyticus in shrimp. Dual luciferase reporter assays demonstrated that LvTRP14 enhanced the transcription factor activity of Relish but attenuated that of Dorsal. Furthermore, silencing of LvTRP14 in vivo had opposite effects on expression of different type of antimicrobial peptides. These suggested that LvTRP14 could play a complex role in shrimp immunity.