Simple SummaryTakifugu obscurusis an economically important fish because of its fast growth rate, tender meat, and high nutritional value. The rapid development of the aquaculture industry in recent years has led to the increasing persistence of hypoxia in aquaculture water systems. Dissolved oxygen is an important factor for fish survival, and low oxygen stress can cause slow growth and reduced immunity of fish. Gills are the main respiratory organs of fish and are greatly affected by changes in dissolved oxygen levels. In this study, the transcriptomic responses ofT. obscurusgill tissues were compared under acute hypoxic stress relative to normoxic and reoxygenated conditions to identify differentially expressed genes associated with hypoxia and understand the adaptive changes of these genes in fish responses to hypoxia. This study provides new insights into the molecular mechanisms underlying the responses ofT. obscurusto hypoxic stress.AbstractTakifugu obscurushas relatively small gills and gill pores, leading to a relatively low respiratory capacity and increased vulnerability to low dissolved oxygen (DO) levels compared to other fish. To investigate the responses ofT. obscurusto acute hypoxic stress, high-throughput-sequencing-based transcriptomic analyses were conducted here to assess the responses ofT. obscurusgills to acute hypoxic stress. Three environmental conditions were compared including normoxia (DO: 7.0 ± 0.2 mg/L), hypoxic stress (DO: 0.9 ± 0.2 mg/L), and reoxygenation (4, 8, 12, and 24 h after return to normoxia) conditions to identify differentially expressed genes (DEGs) responsive to hypoxia. A total of 992, 877, 1561, 1412, and 679 DEGs were identified in the normoxia and reoxygenation for 4, 8, 12, and 24 h groups in comparison to the hypoxia groups, respectively. The DEGs were primarily associated with oxidative stress, growth and development, and immune responses. Further functional annotation enrichment analysis of the DEGs revealed that they were primarily related to cytokine–cytokine interactions, transforming growth factor β receptor (TGF-β), cell adhesion molecules (CAMs), the vascular endothelial growth factor (VEGF) signaling pathway, and the mitogen-activated protein kinase (MAPK) signaling pathway. These results provide new insights into the physiological and biochemical mechanisms ofT. obscurusadaptations to hypoxic stress. Furthermore, these results provide a framework for future studies into the molecular mechanisms of hypoxia tolerance and the healthy culture ofT. obscurusand other fish.