The macrophage migration inhibitory factor (MIF) is an important proinflammatory cytokine that mediates both innate and adaptive immune responses. In this study, we identified a homolog of MIF in the Pacific white shrimp Litopenaeus vannamei. The MIF cDNA contained a 363-bp open reading frame encoding a 120-amino acid protein with a calculated molecular mass of 13.442 kDa and a theoretical isoelectric point of 6.57. The L. vannamei MIF shared high amino acid identity with MIFs of other invertebrates. Tissue distribution analysis by quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the L. vannamei MIF was abundantly expressed in the blood, heart, and hepatopancreas, was moderately expressed in the gill, and was weakly expressed in the muscle and intestine. Furthermore, in order to gain a basic understanding of the role of MIF in the shrimp immune response against viral infection, its mRNA expression was determined in the hepatopancreas of L. vannamei at different stages after white spot syndrome virus (WSSV) challenge using qRT-PCR. The result indicated that the expression of MIF was significantly upregulated after WSSV injection, suggesting that MIF may be involved in the response to viral infection in shrimp.