Background & Aims Insulin resistance is strongly associated with non-alcoholic fatty liver disease, a chronic, obesity-related liver disease. Increased endoplasmic reticulum (ER) stress plays an important role in the development of insulin resistance. In this study, we investigated the roles of miRNAs in regulating ER stress in the liver of rats with obesity. Methods We used miRNA microarray to determine the miRNA expression profiles in the liver of rats fed with a high fat diet (HFD). We used prediction algorithms and luciferase reporter assay to identify the target gene of miRNAs. To overexpress the miRNA miR-30b or inhibit miR-30b rats were injected with lentivirus particles containing PGLV3-miR-30b or PGLV3-miR-30b antimiR through tail vein. Hepatic steatosis was measured using transient elastography in human subjects. Results Our data showed that miR-30b was markedly up-regulated in the liver of HFD-treated rats. Bioinformatic and in vitro and in vivo studies led us to identify sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), as a novel target of miR-30b. Overexpression of miR-30b induced ER stress and insulin resistance in rats fed with normal diet, whereas inhibition of miR-30b by miR-30b antimiR suppressed ER stress and insulin resistance in HFD-treated rats. Finally, our data demonstrated that there was a positive correlation between serum miR-30b levels and hepatic steatosis or homoeostasis model assessment of insulin resistance (HOMA-IR) in human subjects. Conclusions Our findings suggest that miR-30b represents not only a potential target for the treatment of insulin resistance, but also a non-invasive disease biomarker of NAFLD.