Abstract: Purpose Study on the relationship between intrauterine hypoxia and aminoglycoside antibiotics (gentamicin) on the development of inner ear dysplasia in neonatal rats. Methods CIH rat model was established. 24 SD pregnant rats were divided into normal group and hypoxia group with 12 rats in each group and 8 weeks after birth. The two groups were randomly divided into four subgroups.( 12 rats in each group) : (1)Normal offspring group,(2) Normal GM group ,(3) Hypoxia group .(4) Hypoxia GM group Four groups of rats were anesthetized and killed after auditory brainstem response (ABR) was measured. The inner ear was examined by WB、Real-time PCR, methylation and so on. Results: ① Analysis of arterial blood gas in pregnant rats: the level of PaO2 SaO2 in pregnant rats in hypoxia group was significantly lower than that in normal group, and the difference was statistically significant.but the Pa CO2 and pH were significantly lower than those in normal group (P < 0.05). ②The difference was not statistically significant. The results of ABR: compared with other groups, there was significant difference in ABR response threshold between the first 4 group and the other groups (P < 0.01).There was no significant difference in ABR response threshold between group 2 (P > 0 05) and group 1 (P > 0 05), but there was no significant difference between group 2 (P > 0 05) and group 1 (P < 0 05). There was significant difference (P < 0.01). There was no significant difference in ABR response threshold between group 2 and group 3 (P > 0.05), But there was significant difference between the ABR reaction threshold and the first group (P < 0.01). ③Real Time-PCR was used to detect the level of GJB2 mRNA in the liver tissues of the offspring, and the Western Blot was used to detect the expression of Connexin26 protein in the inner ear of the offspring, and the results were consistent. There was a significant difference in the level of GJB2 mRNA between the fourth groups compared with the other groups (P < 0.01). There was no significant difference in the GJB2 mRNA level between the second groups and the third groups (P > 0.05), but there was a significant difference compared with the first groups (P < 0.01). ④Methylation sequencing: GJB2 gene promoter island 1 and island 2 region methylation analysis showed that the percentage of methylation rate in group 4 was significantly higher than that in the other three groups (P < 0.05). There was no difference between group 2 and group 3 (P > 0.05).But compared with the first group, the difference was statistically significant (P < 0.05). Conclusion: The effect of hypoxia and gentamicin in pregnancy can lead to hearing damage and synergistic effect . The methylation level of GJB2 gene promoter is increased , the level of GJB2 mRNA decreases , and the expression of Connexin26 protein decreases