AXL plays crucial roles in the tumorigenesis, progression, and drug resistance of neoplasms; however, the mechanisms associated with AXL overexpression in tumors remain largely unknown. In this study, to investigate these molecular mechanisms, wildtype and mutant proteins of arrestin domain-containing protein 3 (ARRDC3) and AXL were expressed, and co-immunoprecipitation analyses were performed. ARRDC3-deficient cells generated using the CRISPR-Cas9 system were treated with different concentrations of the tyrosine kinase inhibitor sunitinib and subjected to cell biological, molecular, and pharmacological experiments. Furthermore, immunohistochemistry was used to analyze the correlation between ARRDC3 and AXL protein expressions in renal cancer tissue specimens. The experimental results demonstrated that ARRDC3 interacts with AXL to promote AXL ubiquitination and degradation, followed by the negative regulation of downstream signaling mechanisms, including the phosphorylation of protein kinase B and extracellular signal-regulated kinase. Notably, ARRDC3 deficiency decreased the sunitinib sensitivity of clear cell renal cell carcinoma (ccRCC) cells in a manner dependent on the regulation of AXL stability. Overall, our results suggest that ARRDC3 is a negative regulator of AXL and can serve as a novel predictor of sunitinib therapeutic response in patients with ccRCC.