Background: Lenvatinib demonstrated noninferiority to sorafenib in overall survival and a statistically significant increase in progression-free survival in patients with unresectable hepatocellular carcinoma (HCC). However, the development of drug resistance of lenvatinib is becoming common. Methods: Here, we performed the first genome-wide CRISPR/Cas9-based screening on lenvatinib-treated HCC cells to identify essential genes for non-mutational mechanisms related to acquired lenvatinib resistance and/or sensitivity in HCC cells. A genome-scale CRISPR knockout (mGeCKOa) library containing 123,411 single guide RNAs (sgRNAs) was used to identify loss-of-function mutations conferring lenvatinib resistance upon HCC cells. CCK8, wound-healing, transwell and flow cytometry assays were used. Subcutaneous xenotransplantation models were used to investigate the role in vivo. Results: By using genome-wide CRISPR/Cas9 library screening, we identify PAK1, a family member of serine/threonine p21-activating kinases, as a critical driver for lenvatinib resistance and the most-effective genetic suppressor of lenvatinib activity. Analysis of the survival of HCC after hepatic resection revealed that low PAK1 expression shortened overall and tumor-free survival (P < 0.001). Further, loss of PAK1 from HCC cell increased the cytotoxicity of lenvatinib in vivo. Lenvatinib treatment activates series of metabolism, including “Glycolysis/ Gluconeogenesis、Cysteine and methionine metabolism、Selenocompound metabolism、Sphingolipid metabolism、Pyruvate metabolism、Metabolic pathways”. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that loss of PAK1 activates the Glycolysis/ Gluconeogenesis and increase the production related metabolites. Concomitantly, activation of PAK1 reduces ROS level and HCC apoptosis upon Lenvatinib treatment. Lenvatinib treatment activates series of metabolism, including “Glycolysis/ Gluconeogenesis”. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that loss of PAK1 activates the Glycolysis/ Gluconeogenesis and increase the production related metabolites. Concomitantly, activation of PAK1 reduces ROS level and HCC apoptosis upon Lenvatinib treatment. Conclusions: In summary, our results demonstrate that targeting PAK1 is an effective approach to overcome lenvatinib drug resistance in HCC.