Objective To clarify the changes in the microRNA (miRNA) expression profile of rat alveolar lavagefluid-derived exosomes after acute ozone exposure and analyze its potential relationship with lung injury. Methods Adult male Wistar rats were exposed to 0 ppm, 0. 5 ppm, and 2 ppm ozone for 6 h in a gas exposure chamber. After 24 h, the rats were anesthetized, the alveolar lavage fluid was collected, and exosomes were extracted. Differentially expressed miRNAs in exosomes were detected by miRNA-seq technology. miRDB databases were used to predict the target genes of the top 10 differentially expressed miRNAs among the groups, and Gene Ontology function enrichment and KEGG Pathway analyses were performed on the target genes. The differentially expressed miRNAs with high expression abundance and good uniformity were verified by quantitative reverse-transcription PCR ( qRT-PCR). Results Compared with the findings in the 0 ppm group, there were 8 up-regulated and 55 down-regulated miRNAs in the 0. 5 ppm group, and 32 up-regulated and 61 down-regulated miRNAs in the 2 ppm group. Compared with the findings in the 0. 5 ppm group, 90 exosomal miRNAs were up-regulated and 74 were down-regulated in the 2 ppm group. The target genes of top 10 differentially expressed miRNAs were mainly involved in regulating DNA binding, heterocyclic compound binding, and affecting transcription, and were closely related to cancer, MAPK, PIK3-AKT, TNF, ErbB, mitophagy, and FoxO signaling pathways. The qRT-PCR showed that the sequencing result were basically reliable. Conclusions Acute ozone exposure can lead to changes in the expression profile of exosomal miRNA in the alveolar lavage fluid of rats. Differentially expressed miRNAs may be involved in pathological processes, such as lung tissue inflammation and lung injury, caused by ozone exposure.