Tumor necrosis factor receptor-associated factor-6 (TRAF6) is a well-established upstream regulator of the IKK complex, essential for the modulation of the NF-κB (nuclear factor kappa B) signaling pathway. Aberrant activation of TRAF6 has been strongly implicated in the pathogenesis of various cancers, including hepatocellular carcinoma (HCC). The speckle type BTB/POZ protein (SPOP), an E3 ubiquitin ligase substrate-binding adapter, constitutes a significant component of the CUL3/SPOP/RBX1 complex, which is closely linked to tumorigenesis. In this study, we demonstrated that the E3 ubiquitin ligase SPOP shielded TRAF6 from proteasomal degradation, leading to the hyperactivation of the NF-κB pathway. Notably, a liver cancer-associated S119N mutation in SPOP resulted in a failure to mediate the ubiquitination and subsequent degradation of TRAF6. Moreover, both gain-of-function and loss-of-function experiments revealed that SPOP inhibits the proliferation and invasion of HCC cells through the TRAF6-NF-κB axis in vitro and in vivo. Taken together, our findings elucidate the underpinning mechanism by which SPOP negatively regulates the stability of the TRAF6 oncoprotein, thus offering a new therapeutic target for HCC intervention.