AbstractMost cullin-RING ubiquitin ligases (CRLs) form homologous assemblies between a neddylated cullin-RING catalytic module and a variable substrate-binding receptor (for example, an F-box protein). However, the vertebrate-specific CRL7FBXW8 is of interest because it eludes existing models, yet its constituent cullin CUL7 and F-box protein FBXW8 are essential for development, and CUL7 mutations cause 3M syndrome. In this study, cryo-EM and biochemical analyses reveal the CRL7FBXW8 assembly. CUL7’s exclusivity for FBXW8 among all F-box proteins is explained by its unique F-box-independent binding mode. In CRL7FBXW8, the RBX1 (also known as ROC1) RING domain is constrained in an orientation incompatible with binding E2~NEDD8 or E2~ubiquitin intermediates. Accordingly, purified recombinant CRL7FBXW8 lacks auto-neddylation and ubiquitination activities. Instead, our data indicate that CRL7 serves as a substrate receptor linked via SKP1–FBXW8 to a neddylated CUL1–RBX1 catalytic module mediating ubiquitination. The structure reveals a distinctive CRL–CRL partnership, and provides a framework for understanding CUL7 assemblies safeguarding human health.