Occludin is an important tight junction (TJ) protein in pulmonary epithelial cells. In this study, we identified changes in occludin in arsenic-induced lung injury in vivo and in vitro. Upon intratracheal instillation with arsenic trioxide (As₂O₃) at a daily dose of 30 μg/kg for 1 week, levels of occludin mRNA and protein expression decreased significantly in mouse lung tissue. Levels of occludin mRNA and protein expression in BEAS-2B cells were reduced upon exposure to As₂O₃ in a concentration- and time-dependent manner. In addition, exposure to As₂O₃ significantly increased expression of p-p38, p-ERK1/2, p-ELK1, and MLCK in mouse lung tissue and BEAS-2B cells. Treatment with As₂O₃ induced oxidative stress in mouse lung tissue and BEAS-2B cells. In BEAS-2B cells, exposure to As₂O₃ reduced transepithelial resistance, which was partially restored with N-acetyl-cysteine (NAC) treatment. Reduced expression of occludin mRNA and protein induced by As₂O₃ was entirely restored with NAC and resveratrol. However, SB203580, PD98059, and ML-7 partially blocked As₂O₃-induced occludin reduction in BEAS-2B cells. These results indicate that As₂O₃ inhibits occludin expression in vivo and in vitro at least partially via the ROS/ERK/ELK1/MLCK and ROS/p38 MAPK signaling pathways.