Multi-subunit cullin-RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4AMBRA
1 (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4AMBRA
1 is required to disrupt the assembly and attenuate the ligase activity of human CRL5SOCS
3 and HIV-1 CRL5VIF complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4AMBRA
1 modulates interleukin-6/STAT3 signaling and HIV-1 infectivity that are regulated by CRL5SOCS
3 and CRL5VIF, respectively. Thus, by discovering a substrate of CRL4AMBRA
1, ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross-regulation pathway.