This study provides a comprehensive and clear investigation into the critical role of host glutamine metabolism in the replication cycle of Avian Leukosis Virus Subgroup J (ALV-J). We hypothesized that ALV-J reprograms host cell metabolism to support its replication. Using both an HD11 macrophage cell line and a chicken infection model, we demonstrated through qPCR and western blot analysis that ALV-J infection significantly upregulates key genes involved in glutamine metabolism, particularly glutaminase (GLS) and the solute carrier transporters SLC1A5 and SLC38A2. In vitro, pharmacological inhibition of GLS with the specific inhibitor BPTES, or culturing under glutamine-deprived conditions, substantially reduced viral replication (as measured by p27 antigen levels) and progeny virion production (via viral titers). Conversely, supplementing the culture medium with exogenous glutamine significantly enhanced ALV-J propagation in a dose-dependent manner. Consistent with cellular findings, in vivo experiments showed that ALV-J infection enhances glutamine metabolic activity—evidenced by increased enzyme activity and metabolite levels—in target organs such as the bursa of Fabricius and the spleen. Importantly, therapeutic administration of BPTES effectively suppressed viral loads in these tissues and alleviated virus-induced pathological damage. Our results conclusively demonstrate that ALV-J exploits host glutamine metabolism to facilitate its replication and propose that targeting this pathway with inhibitors such as BPTES represents a novel and promising therapeutic strategy against ALV-J infection and associated diseases. Further investigation into the specificity and safety of glutamine-targeting approaches will help advance their translational potential in poultry health.