Abstract: To examine the molecular regulatory mechanism of microRNAs and target genes in Litopenaeus vannamei during cold adaptation, we conducted microRNAs (miRNAs) analysis on the hepatopancreas of L. vannamei under normal temperature of 28℃ and cold acclimation (16℃ for 6 days) by using Solexa sequencing. In total, 10690259 and 8587144 unique sequences of 18—32 nt length were obtained from small RNA libraries at room temperature and low temperature, including 57 and 48 known mature miRNAs, respectively. Expression analysis revealed 25 significantly differential expressed miRNAs between groups, and three of them were further confirmed by PCR. Moreover, we also observed that cold regulated target genes of significantly differential expressed miRNAs including the nuclear export mediator factor Nemf-lik, synapse-associated protein, seleno proteins and DEAD-box RNA helicase Variant 1. These results suggest that miRNAs and their target genes may mediate the cold adaption in L. vannamei.