Human cytomegalovirus (HCMV) UL42 is classified as a CMV-specific but function-unknown gene. According to its amino acid sequence, UL42 has a C-terminal hydrophobic domain predicted to be a transmembrane domain and two PPxY (PY) motifs in its N terminus, but no N-terminal signal peptide. These features resemble those of herpes simplex virus (HSV) UL56 and varicella-zoster virus ORF0. HCMV UL42 interacts with Itch, a member of the Nedd4 family of ubiquitin E3 ligases, through its PY motifs as observed in HSV UL56. HCMV UL42 was partially colocalized with the trans-Golgi network and cytoplasmic vesicles in transfected fibroblasts. Itch was colocalized with HCMV UL42 and accumulated in a fine-speckled pattern in the cytoplasm. UL42 induced the ubiquitination and degradation of Itch in HCMV-infected fibroblasts, and was partially colocalized with p62, a ubiquitin-binding protein, and CD63, a marker of lysosome and multivesicular bodies. The electrophoretic pattern of Itch was altered by infection with HCMV and the amount of Itch was increased by the deletion of UL42. Our findings suggest that the regulatory function of the Nedd4 E3 ligase family and the structural features of HCMV UL42 are conserved characteristics in herpesviruses.