<p>Sulfur mustard (SM), a highly reactive alkylating agent, remains a significant threat to both military personnel and civilian populations due to its toxic effects. Although substantial research has been conducted, the precise apoptotic mechanisms triggered by SM exposure are not yet fully understood. This study aimed to assess potential differences in the apoptosis following SM-induced acute pulmonary injury via the two routes. An acute pulmonary injury model was developed using SM at an equitoxic dose (1 LD₅₀), administered via either a single intraperitoneal injection or intratracheal instillation. Protein expression levels and mRNA expressions of cysteinyl aspartate specific proteinase-2 (caspase-2), caspase-4, caspase-7, caspase-8, p53, and factor-associated suicide ligand (FasL) were evaluated using immunohistochemistry labeling and polymerase chain reaction analysis. At equitoxic doses, the intraperitoneal SM group exhibited significantly higher protein expression ratios and mRNA expression levels of caspase-2, caspase-4, caspase-7, caspase-8, p53, and FasL in the epithelial cells of the alveolar septa from the right lower lobe of the rat’s lungs than the intratracheal SM group. Both protein and mRNA expression levels increased progressively over time in both groups. The findings indicate that SM in early-phase induces apoptosis through intrinsic and extrinsic pathways as well as possible endoplasmic reticulum stress-related pathway. These insights contribute to a deeper understanding of SM toxicity and offer a basis for developing targeted therapeutic strategies to mitigate its harmful effects.</p> Graphical Abstract <p></p>