OBJECTIVES: The natural triterpenoid compound celastrol ameliorates insulin resistance (IR) in animal models, but the underlying molecular mechanism is unclear. In this study, we investigated how celastrol regulates IR. METHODS: The HepG2 cellular IR model was initially established with palmitic acid (PA). The expression and activity of glucose transporter 4 (GLUT4), insulin receptor substrate-1 (IRS1) and 9 microRNAs (miRNAs) (miR-7, -34a, -96, -113, -126, -145, -150, -223 and -370) were detected before and after celastrol treatment using the PA-induced HepG2 IR model. RESULTS: The results showed that 250 µM PA for ≥2 days was optimal for inducing IR in HepG2 cells; 600 nM celastrol significantly attenuated the PA-induced IR in HepG2 cells. The PA-induced GLUT4 and IRS1 downregulation and Ser307 phosphorylation on IRS1 was reversed by subsequent treatment with 600 nM celastrol for 6 h. We next investigated which IR-related miRNAs were possible upstream regulators of celastrol-mediated reversal of PA-induced HepG2 IR. Two miRNAs, miR-150 and -223, were significantly downregulated by PA and were re-raised by subsequent celastrol treatment; and miR-223 was upstream of miR-150. Moreover, knocking down miR-223 abolished celastrol's anti-IR effects in the PA-induced model. CONCLUSIONS: Collectively, our results demonstrated that celastrol reverses PA-induced IR-related alterations, in part via miR-223 in HepG2 cells. Further investigation is warranted for establishing the clinical potential of celastrol in treating IR-related disorders.