Sulfur mustard (HD) is a vesicating and alkylating agent widely used on the battlefield during World War I and more recently in the Iran-Iraq War. It targets the eyes, skin, and lungs, producing skin burns, conjunctivitis, and compromised respiratory function; early acute effects lead to long-term consequences. However, it is the effects on the lungs that drive morbidity and eventual mortality. The temporal postexposure response to HD within lung tissue raises the question of whether toxicity is driven by the alkylating properties of HD on critical homeostatic pathways. We have established an anesthetized swine model of inhaled HD vapor exposure to investigate the toxic effects of HD 12 h postexposure. Large white female swine were anesthetized and instrumented prior to exposure to air, 60 (sublethal) or 100 μg·kg-1 (∼LD40) doses of HD (10 min). Physiological parameters were continuously assessed. Data indicate that exposure to 100 μg·kg-1 HD lowered arterial blood oxygenation and increased shunt fraction and lavage protein compared with those of air-exposed controls and the 60 μg·kg-1 dose of HD. Histopathology showed an increased total pathology score between the 100 μg·kg-1 HD group and air-exposed controls. Principal component analysis of differentially expressed genes demonstrated a distinct and separable response of inhaled HD between air-exposed controls and the 60 and 100 μg·kg-1 doses of HD. Canonical pathway analysis demonstrated changes in acute phase response signaling, aryl hydrocarbon receptor signaling, NRF-2 mediated oxidative stress, and zymosterol biosynthesis in the 60 and 100 μg·kg-1 HD dose group. Transcriptional changes also indicated alterations in immune response, cancer, and cell signaling and metabolism canonical pathways. The 100 μg·kg-1 dose group also showed significant changes in cholesterol biosynthesis. Taken together, exposure to inhaled HD had a significant effect on physiological responses coinciding with acute changes in gene expression and lung histopathology. In addition, transcriptomics support the observed beneficial effects of N-acetyl-l-cysteine for treatment of acute inhalation HD exposure.