Tumor necrosis factor alpha-induced protein 1 (TNFAIP1) modulates a plethora of important biological processes, including tumorigenesis and cancer cell migration. However, the regulatory mechanism of TNFAIP1 degradation remains largely elusive. In the present study, with a label-free quantitative proteomic approach, TNFAIP1 was identified as a novel ubiquitin target of the Cullin-RING E3 ubiquitin ligase (CRL) complex. More importantly, Cul3-ROC1 (CRL3), a subfamily of CRLs, was identified to specifically interact with TNFAIP1 and promote its polyubiquitination and degradation. Mechanistically, BTBD9, a specific adaptor component of CRL3 complex, was further defined to bind and promote the ubiquitination and degradation of TNFAIP1 in cells. As such, downregulation of BTBD9 promoted lung cancer cell migration by upregulating the expression of TNFAIP1, whereas TNFAIP1 deletion abrogated this effect. Finally, bioinformatics and clinical sample analyses revealed that BTBD9 was downregulated while TNFAIP1 was overexpressed in human lung cancer, which was associated with poor overall survival of patients. Taken together, these findings reveal a previously unrecognized mechanism by which the CRL3BTBD9 ubiquitin ligase controls TNFAIP1 degradation to regulate cancer cell migration.