The immediate-early protein, BICP0, of bovine herpesvirus 1 (BHV-1) transactivates a variety of viral and cellular genes. In a yeast two-hybrid cDNA library screening, we found that lipocalin-type prostaglandin D synthase, which catalyzes the production of prostaglandin D2 (PGD2), is a cellular target of BICP0. We observed that, during wild-type BHV-1 infection, PGD2 levels were increased intracellularly and decreased in the medium. These effects were absent upon infection with recombinant BHV-1 expressing β-galactosidase instead of BICP0 (A2G2). Transient-expression assays showed that BICP0 alone caused a significant increase in PGD2 levels in the cell. PGD2 repressed BHV-1 replication in cultured cells. Antiviral activities of prostaglandins have been documented long ago, but their mode of action remains to be clarified. Here we provide evidence that PGD2 impairs the transactivation ability of BICP0 that is necessary for efficient virus replication.