Berberine (BBR), a natural isoquinoline alkaloid exhibiting insulin sensitizing activity, has been applicated in the treatment of diabetes. However, until now, the exact target of BBR has not been well investigated. Here, primary hepatocytes pre-treated with TNF-α were used to evaluate the role of BBR on hepatic insulin sensitivity. Western blot and immunoprecipitation were used to investigate the effect of BBR on the crosstalk between TNF-α pathway and insulin signaling pathway. Molecular docking was used to verify the interactions between BBR and its potential targets. BBR inhibits the MEKK1 and MEK1/2, and thus suppresses the activation of their downstream ERK1/2. It attenuates the ERK1/2-induced serine phosphorylation of IRS-1 and thus enhances IRS-1 tyrosine phosphorylation and Akt activation. By molecular docking, BBR is proved to efficiently bind MEK1/2. MEKK1 is also considered as BBR target for its similarity in primary structure with MEK1/2. In conclusion, BBR ameliorates TNF-α-induced hepatic insulin resistance by targeting MEKK1 and MEK1/2.© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.