Bovine herpesvirus 1 (BoHV-1) productive infection stimulates β-catenin-dependent transcription to facilitate virus replication. Phosphoglycerate kinase 1 (PGK1), which catalyses the initial step of ATP production during glycolysis, also has a mitochondrial form that is implicated in tissue injury across various diseases. However, the relationship between BoHV-1 replication and the PGK1 signalling pathway is not yet fully understood. In this study, we discovered that PGK1 signalling significantly influences BoHV-1 replication, with the virus infection leading to a marked increase in the accumulation of PGK1 proteins in mitochondria. Overexpression of β-catenin reduces PGK1 steady-state protein levels while overexpressing PGK1 boosts β-catenin protein expression-a phenomenon that reverses upon virus infection. Importantly, consistent with PGK1's vital role in virus replication, PGK1 stimulates β-catenin-dependent transcriptional activity, partly by promoting the nuclear accumulation of transcriptionally active β-catenin and phospho-β-catenin (S552) in virus-infected cells. In summary, our findings suggest for the first time that PGK1 signalling may be involved in BoHV-1 replication and contribute to virus pathogenicity.