Human Nedd4-1 (Neuronal precursor cell expressed developmentally down-regulated gene 4-1) is an E3 ubiquitin ligase that negatively regulates the epithelial Na+ channel (ENaC). Nedd4 is composed of a C2 domain, four WW domains and a ubiquitin ligase Hect domain. The WW domains interact with the ENaC subunits via recognition of “PY” motifs. The third WW domain (WW3*) binds the PY motif ENaC cognitive peptide with a Kd of ~30 μM; the highest of all four WW domains. To understand the mechanism of this interaction in detail, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in the free form and in complex with a C-terminal human ENaC α-subunit peptide (PPxYxxL).1 The structure reveals that the peptide interacts in a similar manner to the other WW domain-ENaC peptide structures, and the apo-state is surprisingly well-ordered, adopting a structure that is similar to the peptide-bound state. Dynamic analysis of the WW3* domain revealed that the apo-state and the domain in complex with the EnaC α-subunit peptide have similar order parameter (S2) values. In contrast, relaxation dispersion data and model-free analysis revealed that many residues of the apo-WW3* domain display μs-ms time scale motions. In the complex, two different conformational exchange processes on the μs-ms time scale were identified. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange plays a role in peptide recognition. Thus, structural and dynamic features appear to define the high binding affinity of WW3*. These results should aid the interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.