RBX1/ROC1 is an essential subunit of the largest multiunit Cullin-RING E3 ligase (CRL), which controls the degradation of diverse substrates, thereby regulating numerous cellular processes. Recently, we reported that RBX1 is overexpressed in hepatocellular carcinomas (HCC) and its expression is negatively correlated with patient survival. Moreover, siRNA silencing of RBX1 inhibits the proliferation of liver cancer cells both in vitro and in vivo by inducing CDKN1A/p21-dependent cell senescence. Interestingly, independent of senescence, RBX1 knockdown also triggers an autophagy response, due, at least in part, to the accumulation of the MTOR-inhibitory protein DEPTOR, a recently identified CRL substrate. Biologically, blockage of autophagy significantly enhances the growth-suppressive effect of RBX1 knockdown by triggering massive apoptosis, indicating that the autophagy response upon RBX1 knockdown serves as a survival signal in liver cells. Similar observations were also made in many types of human cancer cells upon inhibition of CRL by MLN4924. These findings suggest that RBX1-CRL is a promising anti-cancer drug target and provide proof-of-concept evidence for a novel drug combination of RBX1-CRL inhibitor and autophagy inhibitor for effective treatment of human cancer.