Viperin is a multifunctional interferon-inducible protein that is directly induced in cells by human cytomegalovirus (HCMV) infection. The viral mitochondrion-localized inhibitor of apoptosis (vMIA) interacts with viperin at the early stages of infection and translocates it from the endoplasmic reticulum to the mitochondria, where viperin modulates the cellular metabolism to increase viral infectivity. Viperin finally relocalizes to the viral assembly compartment (AC) at late stages of infection. Despite the importance of vMIA interactions with viperin during viral infection, their interacting residues are unknown. In the present study, we showed that cysteine residue 44 (Cys44) of vMIA and the N-terminal domain (amino acids [aa] 1 to 42) of viperin are necessary for their interaction and for the mitochondrial localization of viperin. In addition, the N-terminal domain of mouse viperin, which is structurally similar to that of human viperin, interacted with vMIA. This indicates that the structure, rather than the sequence composition, of the N-terminal domain of viperin, is required for the interaction with vMIA. Recombinant HCMV, in which Cys44 of vMIA was replaced by an alanine residue, failed to translocate viperin to the mitochondria at the early stages of infection and inefficiently relocalized it to the AC at late stages of infection, resulting in the impairment of viperin-mediated lipid synthesis and a reduction in viral replication. These data indicate that Cys44 of vMIA is therefore essential for the intracellular trafficking and function of viperin to increase viral replication. Our findings also suggest that the interacting residues of these two proteins are potential therapeutic targets for HCMV-associated diseases. IMPORTANCE Viperin traffics to the endoplasmic reticulum (ER), mitochondria, and viral assembly compartment (AC) during human cytomegalovirus (HCMV) infection. Viperin has antiviral activity at the ER and regulates cellular metabolism at the mitochondria. Here, we show that Cys44 of HCMV vMIA protein and the N-terminal domain (aa 1 to 42) of viperin are necessary for their interaction. Cys44 of vMIA also has a critical role for viperin trafficking from the ER to the AC via the mitochondria during viral infection. Recombinant HCMV expressing a mutant vMIA Cys44 has impaired lipid synthesis and viral infectivity, which are attributed to mislocalization of viperin. Cys44 of vMIA is essential for the trafficking and function of viperin and may be a therapeutic target for HCMV-associated diseases.