Nedd4-family E3 ubiquitin ligases regulate signaling in intracellular pathways that control cancer, blood pressure, iron metabolism, and inflammation. These E3 ligases are catalytically active, and share a highly conserved, modular architecture. How Nedd4 family members are differentially regulated, despite their high degree of homology, is unclear. A regulatory mechanism that maintains an inactive state, common to Nedd4 family members, is autoinhibition. The majority of Nedd4 family members are autoinhibited by an intramolecular interaction between the N terminal C2 domain and the HECT domain. One subfamily of Nedd4 family members that includes the E3 ligase Itch does not appear to be regulated by a C2 domain interaction. The molecular mechanism regulating activation of these E3 ligases is unclear. Here I present findings that illustrate a novel intramolecular interaction regulating Itch and related Nedd4 family member activation that distinguish these E3s as functionally distinct from other Nedd4 family members. I have determined that Itch is autoinhibited by an intramolecular interaction between two central WW domains and two regions in its HECT domain. While capable of interacting with an E2 in its autoinhibited state, Itch is unable to accept ubiquitin onto its catalytic cysteine until this WW domain – HECT domain mediated autoinhibition is relieved. Ndfip proteins bind the WW domains of Itch to release the HECT, facilitating the E2-E3 transthiolation reaction and Itch activation. This inhibitory mechanism can be generalized to the closely related family member WWP2 and, by similarity, WWP1. Further, I tested whether multiple PY motifs are required for Ndfip1 to activate Itch, and found that single PY motifs were not sufficient to relieve Itch autoinhibition. This indicates that activators of Itch can be functionally distinguished from substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 transthiolation, and suggest that Nedd4 family members can be functionally subdivided based on their autoinhibitory mechanisms.