Objective To clarify the changes of microRNA ( miRNA ) expression profile in rat alveolar lavage fluid-derived exosomes after acute ozone exposure, and to 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, alveolar lavage fluid was collected and exosomes were extracted. The differentially expressed miRNAs in exosomes were detected by miRNA-seq technology. The miRDB databases were used to predict the target genes of differentially expressed Top 10 miRNAs between groups, and GO analysis and KEGG analysis were performed on the target genes. The differentially expressed miRNAs with high expression abundance and good uniformity in the group were verified by qRT-PCR. Results Compared with the 0 ppm group, there were 8 up-regulated and 55 down-regulated miRNAs in the 0.5 ppm group, while 32 up-regulated and 61 down-regulated miRNAs in the 2 ppm group. Compared with the 0.5 ppm group, 90 exosomal miRNAs were up-regulated and 74 were down-regulated in the 2 ppm group. The results of enrichment analysis showed that the target genes of differentially expressed Top 10 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 results of qRT-PCR showed that the sequencing results were basically reliable. Conclusion Acute ozone exposure can lead to changes in the expression profile of exosomal miRNA in alveolar lavage fluid of rats. Differential miRNA may be involved in pathological processes such as lung tissue inflammation and lung injury caused by ozone exposure.