Human cytomegalovirus (HCMV) is a ubiquitous virus that is associated with severe morbidity and mortality in immunocompromised individuals and is the major infectious cause of birth defects in the developed world. Interferons (IFNs) are innate immune cytokines known to play a crucial role in controlling cytomegalovirus replication exemplified by the number of virally encoded gene products that limit the production of and/or the action of IFNs. However to our knowledge the effect of disabling the IFN response on HCMV replication has not been previously investigated. The effect of such an abrogation of the interferon response on HCMV infection and replication was examined using primary human fibroblast cell lines expressing either the V protein of Parainfluenza type V or the nPro protein of Bovine Viral Diarrhoea Virus that block the response to or the production of type I IFNs respectively. In these IFN-defective cell lines, although there was no enhancement of the efficiency of initial infection, a number of different HCMV strains (both UL128L+ and UL128L-) produced significantly larger plaques compared to control cells. The virus also spread and replicated more rapidly than in parental fibroblasts demonstrating the key role of IFN-b in controlling viral replication. In addition, these cell lines facilitated the identification of IFN-beta as a potent driver of the upregulation of the immunoregulatory lectin, galectin-9, following HCMV infection. These cell lines demonstrate the vital role of IFNs in controlling HCMV replication and provide useful tools to further investigate the IFN response to HCMV.