As a non-coding RNA, microRNA (miRNA) has been proven to play an important role in the development and progression of type 2 diabetes mellitus (T2DM). Highland barley is a whole grain from the Tibetan areas of China. Our previous studies have demonstrated its hypoglycemic effect. To further explore the underlining molecular mechanism, we investigated the effect of highland barley intervention on liver miRNA expression profiles in diabetic mice. Our results showed that ten differentially expressed miRNA among different groups were identified and their target genes were predicted. Remarkably, many glycometabolism-associated genes, including Foxo3, Nras, Rptor, Igf1r, Tsc2 and Braf, were negatively regulated by miR-122-5p, miR-503-5p, miR-455-5p and miR-210-3p, respectively. Pathway enrichment analysis revealed these target genes were mainly involved in AMPK, MAPK and FOXO signaling pathways. Thereby, these miRNA and mRNA were validated using qRT-PCR, and the results were consistent with the small RNA-seq and expectations. Highland barley could regulate the MAPK, AMPK, and FOXO signaling pathways by regulating critical miRNA-mRNA pairs, e.x. miR-210-3p-Tsc2/Braf, miR-122-5p-Foxo3, and miR-455-5p-Igf1r, thereby improving blood glucose metabolism in diabetic mice. The present study preliminarily explored the hypoglycaemic effects of highland barley based on transcriptomics, and more detailed and in-depth studies on this topic are needed in the future.