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Salinity is a common abiotic stress in the culture of penaeid shrimp. Through post-transcriptional regulation of gene transcripts, microRNAs (miRNAs) play an important role in the adaptation to a stressful environment. However, the involvement of miRNAs in the salinity stress response of shrimp remains unclear. In the present study, the sequence and expression profile of miRNAs in the hepatopancreas of low-salinity-treated Penaeus monodon were obtained by the high-throughput sequencing technique. A total of 679 miRNAs were identified, including 167 miRNAs that were significantly differentially expressed after low-salinity exposure (p < 0.05). Remarkably, most of these miRNAs were downregulated, suggesting that a series of genes were activated to participate in stress response. In addition, 43 miRNAs differentially expressed at all treatment were selected as putative key modulators. Enrichment analysis of genes targeted by these miRNAs indicated that a network that consists of the nervous system, the immune system, and the endocrine system played a crucial role in maintaining the homeostasis of P. monodon under low-salinity stress. These findings may help contribute to a better understanding of the mechanism that regulates salinity tolerance in shrimp and provide valuable genetic information for subsequent studies.