Decapod iridescent virus 1 (DIV1) results in severe economic losses in shrimp aquaculture. However, little is known about the physiological effect of DIV1 infection on the host. In this study, we found that the lethal dose 50 of DIV1-infected
Litopenaeus vannamei
after 48, 72, 96, and 156 h were 4.86 × 10
6
, 5.07 × 10
5
, 2.13 × 10
5
, and 2.38 × 10
4
copies/μg DNA, respectively. In order to investigate the mechanisms of DIV1 infection, a comparative transcriptome analysis of hemocytes from
L. vannamei
, infected or not with DIV1, was conducted. The BUSCO analysis showed that the transcriptome was with high completeness (complete single-copy BUSCOs: 57.3%, complete duplicated BUSCOs: 41.1%, fragmentation: 0.8%, missing: 0.8%). A total of 168,854 unigenes were assembled, with an average length of 601 bp. Based on homology searches, Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and cluster of orthologous groups of proteins (KOG) analysis, 62,270 (36.88%) unigenes were annotated. Among them, 1,112 differentially expressed genes (DEGs) were identified, of which 889 genes were up-regulated and 223 genes were down-regulated after DIV1 infection. These genes were mainly annotated to the major metabolic processes such as fructose and mannose metabolism, carbon metabolism, and inositol phosphate metabolism. Among these metabolic pathways, the triosephosphate isomerase (
TPI
) family was the most eye-catching DEG as it participates in several metabolic processes. Three types of
TPI, LvTPI-like, LvTPI-Blike
, and
LvTPI-Blike1
, were obtained for gene silencing by RNA interference. The results showed that
LvTPI-like
and
LvTPI-Blike1
silencing caused a high mortality rate among
L. vannamei
. However,
LvTPI-like
and
LvTPI-Blike
silencing reduced DIV1 replication in DIV1-infected
L. vannamei
. All the results indicated that
TPI-like
genes play an important role during DIV1 infection, which provides valuable insight into the infection mechanism of DIV1 in shrimp and may aid in preventing viral diseases in shrimp culture.