Objective To investigate the effects of geniposide on hearing loss and nuclear transcription factor E2 related factor 2/ heme oxygenase-1 (Nrf2/HO-1) signaling pathway in rats with acute otitis media (AOM).Methods AOM rat model was constructed, and the experimental rats were randomly divided into control group, AOM group, Geni low-dose, medium-dose, and high-dose groups (Geni-L, Geni-M, Geni-H groups), and Geni high-dose + pathway inhibitor ML385 group (Geni-H+ML385 group). Rats in each group were subjected to hearing tests. Enzyme-linked immunosorbent assay was applied to detect serum inflammatory factors and oxidative stress level. HE staining was applied to observe pathological damage of ear mucosal tissue. Western blot was applied to detect the expressions of Nrf2/HO-1 signaling pathway related proteins.Results Compared with the control group, the middle ear mucosa in the AOM group was disorganized, with obvious swelling and shedding of ear mucosal cells, significant infiltration of inflammatory cells and proliferation of fibrous tissue, and significantly thickened mucosal thickness. Meanwhile, the auditory brainstem response threshold, levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), and expression of Kelch-like epichlorohydrin-associated protein-1 (KEAP1) were elevated, the activities of superoxide dismutase (SOD) and glutathione (GSH), and the expression of Nrf2 and HO-1 were reduced (P<0.05). Compared with the AOM group, the pathological symptoms of the middle ear mucosal tissue in the Geni-L, Geni-M, and Geni-H groups were reduced, the degree of mucosal swelling and inflammatory cell infiltration were reduced, the mucosal thickness became thinner, and the ABR response threshold, levels of IL-1β, IL-6, TNF-α, MDA and expression of KEAP1 were decreased, the activities of SOD and GSH and the expressions of Nrf2 and HO-1 were increased, with the most obvious changes observed in the Geni-H group (P<0.05). The pathological symptoms of the middle ear mucosal tissue in the Geni-H+ML385 group were aggravated compared to the Geni-H group, with obvious swelling and shedding of ear mucosal cells, increased inflammatory cell infiltration and fibrous tissue proliferation and increased mucosal thickness, the ABR response threshold, levels of IL-1β, IL-6, TNF-α, MDA and expression of KEAP1 were elevated, the activities of SOD and GSH, and the expressions of Nrf2 and HO-1 were reduced (P<0.05).Conclusion Geniposide can improve hearing loss in AOM rats, and its mechanism is related to the activation of the Nrf2/HO-1 signaling pathway.