In biological evolution, gene duplication (GD) generates new genes to facilitate new functions. C-type lectins (CTLs) in crayfish have been extended by GD to expand their family members. In this study, four CTL genes generated by GD were identified from Procambarus clarkii (PcLec1–4). Among these four genes, PcLec1 can also generate new isoforms with different numbers of tandem repeats through DNA slip mispairing. PcLec1–4 was widely expressed in multiple tissues. The expression levels of PcLec1–4 were upregulated in the intestine of P. clarkii upon white spot syndrome virus (WSSV) challenge at multiple time points. Further analysis indicated that GATA transcription factor regulated PcLec1–4 expression. RNA interference and recombinant PcLec1–4 protein injection experiments suggested that PcLec1–4 promoted the expression of calreticulin (PcCRT) and negatively regulated the expression of antimicrobial peptides, thereby promoting WSSV replication. This study contributes to the understanding of the function of CTLs produced by GD during WSSV invasion in crustaceans. •Four CTL genes were identified from Procambarus clarkii generated by gene duplication.•The transcription factor GATA controls the expression of PcLec1-4.•PcLec1-4 promotes viral replication through regulation of Pccalreticulin and antimicrobial peptides, respectively.