Human exploration of the Moon and Mars will inevitably result in exposure to extraterrestrial dust. We investigated the potential pulmonary toxicity of JSC Mars-1(Martian dust simulant, respirable diameter 1.45 μm) using an advanced multicellular lung mucosa model with dust applied apically to human bronchial epithelial cells cultured at the air-liquid interface, with doses 55, 222, and 890 μg/cm2, PBS (sham). Compared to the sham, medium and high doses of JSC Mars-1 increased necrosis-related gene HMGB1 expression. The cellular total reactive oxygen species (ROS) levels increased in a dose-dependent manner. The expression of antioxidant-related genes, HMOX-1 and SOD3, increased at all dose levels. Interleukin-6 (IL-6) protein and gene expression increased, and tumor necrosis factor alpha (TNF-α) after high dose. CXCL8 mRNA was elevated at medium and high doses. TLR4 surface and gene expression decreased at medium and high doses. JSC Mars-1 dust increased cytotoxicity, oxidative stress, and immune responses suggesting potential pulmonary toxic effects, providing insight to molecular mechanisms behind potential adverse respiratory effects.