Fish often faces exposures to acute environmental hypoxia either spatially, such as moving through a water column, or temporally due to the seasonal changes in eutrophication. The initial response for most fish in general increases total gill ventilation in low dissolved oxygen environments. However, few studies focus on the molecular regulation mechanisms of gills under acute hypoxia stress. In this study, we investigated the transcriptomic response in the gill tissues of a hypoxia tolerant fish, Nile tilapia Oreochromis niloticus, in response to 12h treatment of acute hypoxia through RNA sequencing (RNA-Seq). We sequenced messenger RNA from 3 control samples and 3 hypoxia treated samples. Bioinformatics analysis identified 239 differentially expressed genes (DEG) and 34 unique genes that had significant differential splicing events in at least one exonic region in gills under acute hypoxia. The spatio-temporal expression analysis in 5 tissues (heart, liver, brain, gill and spleen) sampled at three time points (6h, 12h, and 24h) under hypoxia treatment disclosed the significant association of differential exon usages in two genes (TLDC2 and SSX2IPA) of our dataset with hypoxia conditions. Further function analysis suggests several energy and immune response-related pathways, e.g., metabolic pathway and Antigen processing and presentation, contained the most abundant DEG genes. We found some GO biological process for DEG genes were significantly enriched under hypoxia stress, such as glycolysis (16.53 fold P = 0.03), metabolic process (1.54 fold P = 0.002), generation of precursor metabolites and energy (5.9 fold P = 0.02), cholesterol metabolic process (10.5 fold P = 0.03), steroid metabolic process (8.46 fold P = 0.0004) and lipid metabolic process (3.79 fold P = 0.01). Our findings suggest abundant differential gene expression changes and alternative splicing events in genes involved in the hypoxia response in gills. Our results provide a basis for exploring the gene regulation mechanism under hypoxia stress and may serve as a resource for performing molecular selective breeding of hypoxia resistant lines in fish.