Berberine (BBR) is an isoquinoline alkaloid found in different plant families such as Berberidaceae, Ranunculaceae, and Papaveraceae. BBR is well-known for its anti-inflammatory, lipid-modifying, anticancer, anti-diabetic, antibacterial, antiparasitic and fungicide activities. Multiple pharmacological actions of BBR stem from different molecular targets of this phytochemical. MicroRNAs (miRs) are single-stranded, evolutionary conserved, small non-coding RNA molecules with a length of 19-23 nucleotides that are involved in RNA silencing and post-transcriptional regulation of gene expression through binding to the 3'-untranslated region (3'UTR) of target mRNA. MiRs emerged as important regulatory elements in almost all biological processes like cell proliferation, apoptosis, differentiation and organogenesis, and numerous human diseases such as cancer and diabetes. BBR was shown to regulate the expression of miRs in several diseases. Here, we reviewed the target miRs of BBR and the relevance of their modulation for the potential treatment of serious human diseases like multiple myeloma, hepatocellular carcinoma, colorectal cancer, gastric cancer, ovarian cancer and glioblastoma. The role of miR regulation in the putative anti-diabetic effects of BBR is discussed, as well.