Duck plague, caused by a highly contagious α-herpesvirus, poses a major threat to waterfowl farming. Although UL50 homologs have been studied in mammalian α-herpesviruses such as HSV-1 and PRV, their role in avian herpesviruses remains unknown. Here, we investigated the function of the DEV UL50 gene, which encodes a conserved viral dUTPase, using bioinformatics, molecular biology, and virological approaches. Sequence analysis confirmed that DEV UL50 retains conserved catalytic motifs and structural features characteristic of α-herpesvirus homologs. A UL50-deleted mutant (ΔUL50) was constructed using the Red recombination system. In vitro, ΔUL50 exhibited reduced replication efficiency in DEFs, characterized by smaller plaques and lower viral titers, although overall growth kinetics were broadly similar to the WT. Notably, in duck DRG neurons, ΔUL50 replication was nearly abolished. GFP-reporter BAC viruses further confirmed that ΔUL50 failed to spread in DRG neurons but retained propagation in DEFs, indicating a cell-type-dependent replication defect. In vivo, ΔUL50 displayed markedly reduced viral loads and attenuated virulence, with no mortality and milder clinical and histopathological changes. These findings demonstrate that UL50 is dispensable but replication-supportive, particularly in non-dividing neuronal cells, highlighting its role in DEV pathogenicity and extending understanding of α-herpesvirus biology beyond mammalian systems.