Objective To investigate the regulation and mechanism of microRNA (miR)-146a on mitochondrial dysfunction in rats with acute respiratory distress syndrome (ARDS). Methods A total of 40 SD rats were divided into control group, model group, agomir NC group and miR-146a agomir group according to random number table method, with 10 rats in each group. The ARDS rat model was constructed by injecting oleic acid into femoral vein of rats in the other 3 groups except the control group. Agomir negative control (NC) or miR-146a agomir was injected into the tail vein after modeling. Alveolar lavage fluid (BALF) was collected in each group, the expression levels of ARDS related miRNAs in BALF was determined by RT-qPCR. The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the BALF supernatant of each group were determined by enzym-linked immunosorbent assay (ELISA), the pathological morphology of lung tissue was observed by hematoxylin-eosin (HE) staining, the ultrastructure of mitochondria was observed by transmission electron microscope, the content of adenosine triphosphate (ATP) in lung tissues was determined by colorimetry, the expressions of optic atrophy protein 1 (Opa1), mitotic fusion protein 2 (Mfn2), dynamic-associated protein 1 (Drp1) and mitochondrial mitogen protein 1 (Fis1) in lung tissues of rats in each group were determined by Western blot, the mRNA expression of miR-146a and tumor necrosis factor receptor-associated protein 6 (TRAF6) in lung tissues of rats in each group were detected by Real-time fluorescence quantitative PCR (RT-qPCR). The bioinformatics online software Targetscan predicted the targeted binding sites of miR-146a and TRAF6, and verified the relationship between the two by double luciferase gene reporting experiment. Results Compared with the control group, in model group rats, the expression levels of miR-7a-5p, miR-138-5p and miR-21-5p in BALF were significantly increased (P<0.05), while the expression levels of miR-17-5p, miR-146a, miR-223-3p and miR-9a-5p were significantly decreased (P<0.05). Among them, miR-146a was significantly decreased (P<0.05). Compared with the control group, the contents of IL-1β, IL-6 and TNF-α in BALF in model group were significantly increased (P<0.05), the inflammatory cell infiltration in lung tissue, alveolar wall thickening, interstitial edema, mitochondrial number decreasing, swelling and ridge breaking or disappearing, ATP content was significantly decreased (P<0.05), the protein expressions of Opa1 and Mfn2 were significantly down-regulated (P<0.05), the protein expressions of Drp1 and Fis1 were significantly up-regulated (P<0.05), the expression of miR-146a was significantly down-regulated (P<0.05), and the mRNA expression of TRAF6 was significantly up-regulated (P<0.05). Compared with model group and agomir NC group, the contents of IL-1β, IL-6 and TNF-α in BALF of rats in miR-146a agomir group were significantly decreased (P<0.05), the inflammatory cell infiltration, alveolar wall thickness and edema in lung tissue were significantly improved, and the number of mitochondria increased and their morphology tended to be normal, ATP content was significantly increased (P<0.05), the protein expressions of Opa1 and Mfn2 were significantly up-regulated (P<0.05), the protein expressions of Drp1 and Fis1 were significantly down-regulated (P<0.05), and the expression of miR-146a was significantly up-regulated (P<0.05), TRAF6 mRNA expression was significantly down-regulated (P<0.05). In addition, there were targeted binding sites in the 3'-UTR region of miR-146a and TRAF6, and the relative luciferase activity of miR-146a mimic was significantly decreased in HEK-293T cells transfected with WT-TRAF6 compared with mimic NC (P<0.05). Conclusion miR-146a plays a protective role in lung tissue by targeting TRAF6 expression, reducing lung tissue injury and improving mitochondrial function in ARDS rats