Nitrogen mustard (NM) is a vesicant that causes lung injury and fibrosis, accompanied by a persistent macrophage inflammatory response. In these studies we analyzed the spleen as a source of these cells. Splenectomized (SPX) and sham control rats were treated intratracheally with NM (0.125 mg/kg) or PBS control. Macrophage responses were analyzed 1–7 days later. Splenectomy resulted in an increase in lung macrophages expressing CCR2, but a decrease in ATR-1α⁺ cells, receptors important in bone marrow and spleen monocyte trafficking, respectively. Splenectomy was also associated with an increase in proinflammatory M1 (iNOS⁺, CD11b⁺CD43⁺) macrophages in lungs of NM-treated rats, as well as greater upregulation of iNOS and COX-2 mRNA expression. Conversely, a decrease in CD11b⁺CD43⁻ M2 macrophages was observed in SPX rats, with no changes in CD68⁺, CD163⁺, CD206⁺, or YM-1⁺ M2 macrophages, suggesting distinct origins of M2 subpopulations responding to NM. Macrophage expression of M2 genes including IL-10, ApoE, PTX-2, PTX-3, 5-HT2α, and 5-HT7 was also reduced in NM-treated SPX rats compared with shams, indicating impaired M2 activity. Changes in lung macrophages responding to NM as a consequence of splenectomy were correlated with exacerbated tissue injury and more rapid fibrogenesis. These data demonstrate that the spleen is a source of a subset of M2 macrophages with anti-inflammatory activity; moreover, in their absence, proinflammatory/cytotoxic M1 macrophages predominate in the lung, resulting in heightened pathology. Understanding the origin of macrophages and characterizing their phenotype after vesicant exposure may lead to more targeted therapeutics aimed at reducing toxicity and disease pathogenesis.