Anthropogenically induced hypoxia in water bodies has been a stressor for fish for many years and is expected to persist in the future. In order to investigate the acclimation response of fish gills to chronic intermittent hypoxia (CIH) stress, we conducted a study using largemouth bass (Micropterus salmoides) exposed to intermittent hypoxia (dissolved oxygen level: 2.0 mg·L− 1) for either one or three hours per day, over a period of 8 weeks. Our findings indicate that exposure to CIH induced remodeling of the gills and an increase in gill surface area. We also observed significant up-regulation of genes related to glycolysis (fba, pgam1, pepck, atp-pfk, pfk-2, g6pi, gapd-1, and pk), while genes associated with cholesterol synthesis (3β-hsd, cyp51, dsdr-x1, dsdr, and dhcr7) were down-regulated following CIH exposure. Furthermore, we observed the presence of elongated megamitochondria in mitochondria-rich cells within the gills of fish exposed to hypoxia. Additionally, numerous genes involved in calcium signaling pathways were up-regulated in the gills of largemouth bass, suggesting an enhanced sensitivity of gills to environmental cues in hypoxia conditions. However, the expression levels of certain genes related to innate and adaptive immune responses were inhibited following CIH exposure. Moreover, the number of mucous cells decreased, potentially making the gills more susceptible to pathogen infections. These findings highlight the potential vulnerability of gills to pathogenic organisms in the presence of CIH. Overall, our study contributes to a better understanding of how fish acclimate to CIH.