This study investigated the acute toxicity of different concentrations of arsenic trioxide (As2O3; ATO) on rat lungs. In total, 160 Wistar rats were randomly divided into the control, low‐, medium‐ and high‐dose groups, which were exposed to 0, 0.16, 1.60 and 16 μg/kg of ATO by intratracheal instillation, respectively. Samples were collected at 6, 12, 24, 48 and 72 hours after exposure and the dynamic changes indicative of acute lung toxicity were monitored. Compared with the control group, the exposure groups exhibited significant changes such as increased lung water content ratio and protein concentration in the bronchoalveolar lavage fluid, pulmonary interstitial thickening, cell membrane edema, increased inflammatory factor concentration, JNK and P38 were significantly activated, and the degree of phosphorylation was increased. Furthermore, all the changes in the exposure groups were exposure concentration‐dependent. ATO respiratory tract exposure can cause restrictive ventilatory disturbance in rats, and the degree of injury is exposure concentration‐dependent.