Aluminum oxide nanoparticles (Al2O3 NPs) are among the most widely used nanomaterials; however, relatively little information about their risk identification and assessment is available. In the present study, we aimed to investigate the potential toxicity of Al2O3 NPs following repeated inhalation exposure in male Sprague-Dawley rats. Rats were exposed to Al2O3 NPs for 28 days (5 days/week) at doses of 0, 0.2, 1, and 5 mg/m3 using a nose-only inhalation system. During the experimental period, we evaluated the clinical signs, body weight change, hematological and serum biochemical parameters, necropsy findings, organ weight, and histopathology findings. Additionally, we analyzed the bronchoalveolar lavage fluid (BALF), including differential leukocyte counts, and aluminum contents in the major organs and blood. Aluminum contents were the highest in lung tissues and showed a dose-dependent relationship in the exposure group. Histopathology showed alveolar macrophage accumulation in the lungs of rats in the 5 mg/m3 group during exposure and recovery. These changes tended to increase at the end of the recovery period. In the BALF analysis, total cell and neutrophil counts and lactate dehydrogenase, tumor necrosis factor-α, and interleukin-6 levels significantly increased in the 1 and 5 mg/m3 groups during exposure. Under the present experimental conditions, we suggested that the no-observed-adverse-effect level of Al2O3 NPs in male rats was 1 mg/m3, and the target organ was the lung.