Human Cytomegalovirus (HCMV) is a ubiquitous, species specific beta herpesvirus that can cause severe systemic disease in immunocompromised and immunonaive individuals. Nuclear Domain 10 (ND10) consists of interchromosomal multiprotein aggregates (also known as PML bodies) that play an essential role in the intrinsic anti-viral response to herpesviruses by restricting viral gene expression. Type I interferons are known to upregulate several ND10 components. The focus of this study has been the regulation of key ND10 component (PML, Sp100 and hDaxx) expression by the HCMV induced interferon response. Investigation of ND10 transcript regulation in primary human fibroblasts (HFs) indicates that PML and Sp100 are upregulated by HCMV infection whilst hDaxx is not. This upregulation did not require de novo viral gene expression and could be demonstrated using supernatants from infected HFs, suggesting that a soluble factor(s) is responsible. Cell lines engineered to block the production of, or the response to, type I interferons were infected with HCMV or treated with supernatants from infected HFs. Upregulation of PML and Sp100 was not detected following HCMV infection in both cell lines, however, treatment with infected HF supernatant enhanced PML and Sp100 transcription in cells with abrogated interferon production. Interferon beta (IFN-b) neutralising antibody administered with infection or to supernatants from infected cells inhibited upregulation of PML and Sp100. To determine the implication of these findings on the infectious ability of HCMV, cells engineered using shRNA to have triple knockdown of the ND10 components PML, Sp100 and hDaxx were analysed. These ND10 deficient cells were significantly more permissive to HCMV infection and, in contrast to control cells, could be productively infected by HCMV even in the presence of IFN-β pre-treatment. Together these data provide evidence that IFN-β can mediate its antiviral activity through control of ND10 expression.