<p>Lenvatinib is the primary targeted drug for hepatocellular carcinoma(HCC). However, the development of drug resistance significantly hinders its therapeutic efficacy. The present study aimed to identify new target molecules of lenvatinib-resistant HCC, and improve the treatment of liver cancer. A lenvatinib-resistant HCC cell line, Bel7404(named Bel7404-R), was established, and the protein expression profile of the Bel7404-R cell line and changes in functional enrichment were analyzed. The expression of the identified lenvatinib-resistant critical protein B4GALT4 was validated, and its correlation with immune infiltration and drug resistance was analyzed. Apoptosis and ferroptosis of HCC cell were observe by Calcein-AM/PI staining and transmission electron microscopy. The results indicated that Bel7404-R cells significantly enhanced colony formation and decreased apoptosis ratio compared with parental Bel7404 cells. 111 upregulated and 170 downregulated proteins in the Bel7404-R cells. Notably, upregulated proteins included B4GALT4, CD55, RFTN1, and SHROOM3, whereas downregulated proteins included GRHPR, CLU, TPM2, TMEM185B and TRPM2. B4GALT4 was significantly overexpressed in Bel7404-R cells and identified as a central protein in the molecular regulatory network of these cells. Knockdown of B4GALT4 expression inhibited the levels of PKM2, LDHA and PI3K/AKT signaling, while stimulating the expression of cleaved-Caspase-3. B4GALT4 could protect the integrity of mitochondria and inhibit ferroptosis. The protein expression profile of Bel7404-R cells were significantly different from those of the parental HCC cells. B4GALT4 was identified as a critical molecule for HCC resisting to lenvatinib, and B4GALT4 can be used as a new therapeutic target for lenvatinib-resistant liver cancer.</p> Graphical Abstract <p></p>