Asparaginase (Elspar) is used in the treatment of acute lymphoblastic leukemia. It depletes plasma asparagine and glutamine, killing leukemic lymphoblasts but also causing immunosuppression. The objective of this work was to assess whether supplementing the diet with glutamine modifies the effect of asparaginase on normal lymphocyte populations in the spleen, thymus, and bone marrow. Mice consuming water ad libitum with or without alanyl-glutamine dipeptide (AlaGln; 0.05 mol/L) were injected once daily with 0 or 3 international units/g body weight Escherichia coli L-asparaginase for 7 d. Tissue expression of specific immune cell surface markers was analyzed by flow cytometry. Asparaginase reduced B220+ and sIgM+ cells in the bone marrow (P < 0.05) and diminished total cell numbers in thymus (-42%) and spleen (-53%) (P < 0.05). In thymus, asparaginase depleted double positive (CD4+ CD8+) and single positive (CD4+ CD8-, CD4-CD8+) thymocytes by over 40% (P < 0.05). In spleen, asparaginase reduced CD19+ B cells to 33% of controls and substantially depleted the CD4+ and CD8+ T cell populations. CD11b-expressing leukocytes were reduced by 50% (P < 0.05). Consumption of AlaGln did not lessen the effects of asparaginase in bone marrow or thymus but mitigated cellular losses in the CD4+, CD8+, and CD11b+ populations in spleen. AlaGln also blunted the increase in eukaryotic initiation factor 2 (eIF2) phosphorylation by asparaginase in spleen, whereas eIF2 phosphorylation did not change in thymus in response to asparaginase or AlaGln. In conclusion, asparaginase reduces maturing populations of normal B and T cells in thymus, bone marrow, and spleen. Oral consumption of AlaGln mitigates metabolic stress in spleen, supporting the peripheral immune system and cell-mediated immunity during asparaginase chemotherapy.