AbstractAccumulating evidence shows HOOK1 disordered in human malignancies. However, the clinicopathological and biological significance of HOOK1 in renal cell carcinoma (RCC) remains rarely studied. In this study, the authors demonstrate that HOOK1 is downregulated in RCC samples with predicted poorer clinical prognosis. Mechanistically, HOOK1 inhibits tumor growth and metastasis via canonical TGF‐β/ALK5/p‐Smad3 and non‐canonical TGF‐β/MEK/ERK/c‐Myc pathway. At the same time, HOOK1 inhibits RCC angiogenesis and sunitinib resistance by promoting degradation of TNFSF13B through the ubiquitin‐proteasome pathway. In addition, HOOK1 is transcriptionally regulated by nuclear factor E2F3 in VHL dependent manner. Notably, an agonist of HOOK1, meletin, is screened and it shows antitumor activity more effectively when combined with sunitinib or nivolumab than it is used alone. The findings reveal a pivotal role of HOOK1 in anti‐cancer treatment, and identify a novel therapeutic strategy for renal cell carcinoma.