Chronic exposure of pancreatic β-cells to saturated fatty acid (palmitic acid or stearic acid) is a leading cause of impaired insulin secretion. However, the molecular mechanisms underlying stearic acid-induced β-cell dysfunction remain poorly understood. Emerging evidence indicates that microRNAs (miRNAs) are involved in various biological functions. The aim of this study was to explore the differential expression of miRNAs and mRNAs, specifically in stearic acid-treated relative to palmitic acid-treated β-cells, and to establish their co-expression networks. β-TC-6 cells were treated with stearic acid, palmitic acid or normal medium for 24 hours. Differentially expressed miRNAs and mRNAs were identified by high-throughput sequencing and bioinformatic analysis. Co-expression network, gene ontology (GO) and pathway analyses were then conducted. Real-time PCR was used to verify changes in the expression of selected miRNAs and mRNAs in β-TC-6 cells and mouse islets. Sequencing analysis detected 656 known and 1729 novel miRNAs. miRNA-mRNA network and Venn diagram analysis yielded two differentially expressed miRNAs and 63 mRNAs. The two miRNAs were confirmed to be differentially expressed specifically in stearic acid-treated cells, in which miR-374c-5p was up-regulated by a 1.801 log2(fold-change) and miR-297b-5p was down-regulated by a -4.669 log2(fold-change). Among the 63 differentially expressed mRNAs, 39 were up-regulated and 24 were down-regulated. This study indicates that expression changes of specific miRNAs and mRNAs may contribute to stearic acid-induced β-cell dysfunction, which provides a preliminary basis for further functional and molecular mechanism studies of stearic acid-induced β-cell dysfunction in the development of type 2 diabetes.