AbstractA significant hurdle in treating glioblastoma (GBM) is addressing the development of drug resistance. In this study, the role of Family of Sequence Similarity 20, Member C (Fam20C) as a central player in bevacizumab resistant GBM mouse model is investigated. In vivo analyses confirm that Fam20C upregulation accelerates drug resistance and correlates with tumor progression. Proteomic analyses of conditioned media and cell lysates subsequent to Fam20C knockout (KO) in GBM cells reveal the regulatory role of Fam20C in both intracellular and extracellular aspects of epithelial‐mesenchymal transition (EMT) and genes associated with AKT signaling. Moreover, In vitro experiments demonstrate that Fam20C activates the AKT signaling pathway, promoting cell proliferation. Elevated levels of Fam20C are observed in human GBM, particularly in the mesenchymal subtype, which correlates with diminished survival rates and increased resistance to various drugs, including temozolomide (TMZ), bevacizumab, epidermal growth factor receptor (EGFR) inhibitors, and other antibody‐based drugs. Notably, even in cases of resistance to gefitinib and hepatocyte growth factor (HGF) antibodies, Fam20C expression is elevated. These findings highlight the pivotal role of Fam20C in driving drug resistance in GBM, suggesting it as a promising target for combination therapies aimed at surmounting this formidable resistance barrier.