Berberine is a Chinese herbal medicine extracted from rhizoma coptidis that functions to improve insulin resistance, hyperlipidemia, hepatosteatosis and inflammation. Berberine can modify the activity of cell metabolism and signaling pathways by regulating expression of genes. However, the roles and effects of differential microRNA (miRNA) expression induced by berberine treatment are largely unexplored. It is believed that miRNAs expression modified by berberine contributes to its therapeutic effects to diseases such as hepatosteatosis and non-alcoholic fatty liver disease. By identifying novel miRNAs and their putative gene targets associated with abnormal hepatic lipid deposition, the underlying mechanism of these diseases could be established and effective therapies against the diseases could be developed. Here, we used the immortalized hepatocyte cell line MIHA as a model to study the effect of berberine on global miRNA expression profile of hepatocytes. Global miRNA expression levels were measured in berberine-treated MIHA cells by quantitative reverse transcription PCR miRNA panel, and the potential berberine regulated miRNAs were then validated in MIHA and HepG2 cells. MicroRNA-373 (MiR-373) was consistently upregulated in both cell lines upon berberine treatments. Gene expression microarray showed that berberine upregulated Early Growth Response 1 (EGR1) level which functioned to transactivate miR-373 expression. Subsequently, we showed that upregulation of miR-373 depleted its target gene AKT serine/threonine kinase 1 (AKT1) mRNA level, which led to the inhibition of AKT-mTOR-S6K signaling pathway in hepatocytes that was critical in the development of hepatosteatosis. Study of the therapeutic effect of manipulating miR-373 against abnormal lipid deposition in liver is warranted.