Human cytomegalovirus (HCMV) is a large double stranded DNA virus of the herpesvirus family. While HCMV infection is generally asymptomatic in the immunocompetent, HCMV can have devastating consequences in immunocompromised individuals, including transplant recipients and neonates. Galectins are a family of cellular proteins characterised by the presence of a carbohydrate recognition domain. Members of this widely expressed protein family have been demonstrated to exert profound effects on host-pathogen interactions. Two of the best studied galectins, galectin-1 (Gal-1) and galectin-9 (Gal-9), have been implicated in a number of immune regulatory processes, and specific roles these for galectins in modulating both anti-viral immunity and regulating direct host-virus interactions have also recently emerged. Previous studies from the Slobedman laboratory have established the differential regulation of Gal-1 and Gal-9 during HCMV infection of primary human fibroblasts. Whilst HCMV infection did not alter total cellular Gal-1 protein levels, expression of cell-surface Gal-1 was significantly upregulated. Conversely, Gal-9 was potently upregulated at the total protein level by a soluble factor, identified as the anti-viral cytokine, IFN-β. The potential for these galectins to directly modulate infection has not previously been studied in the context of HCMV. Infection studies utilising recombinant protein treatment, revealed Gal-9, but not Gal-1, as an anti-viral lectin that potently inhibited HCMV infection. Further analysis established that Gal-9 mediated inhibition of infection was carbohydrate recognition domain-dependent and blocked by anti-Gal-9 specific antibodies. Temperature-shift studies that separated the binding and entry stages of infection, identified Gal-9 specific inhibition as mediated primarily at the level of viral entry rather than binding, and appeared to be dependent on binding to the virion rather than interacting with cellular ligands. This study provides the first evidence of a novel role for Gal-9 as an anti-viral, IFN-β induced protein, that exerts a profound inhibitory effect on HCMV infection.