Identification of differentially expressed genes (DEGs) and regulated pathways in response to stressors using a whole-genome approach is critical to understanding the mechanisms underlying stress responses. We challenged Asian seabass with lipopolysaccharide (LPS), Vibrio harveyi, high salinity and fasting, and sequenced six cDNA libraries of intestine samples using Roche 454 RNA-seq. Over 1 million reads (average size: 516 bp) were obtained. The de novo assembly obtained 83 911 unisequences with an average length of 747 bp. In total, 62.3% of the unisequences were annotated. We observed overall similar expression profiles among different challenges, while a number of DEGs and regulated pathways were identified under specific challenges. More than 1000 DEGs and over 200 regulated pathways for each stressor were identified. Thirty-seven genes were differentially expressed in response to all challenges. Our data suggest that there is a global coordination and fine-tuning of gene regulation during different challenges. In addition, we detected dramatic immune responses in intestines under different stressors. This study is the first step towards the comprehensive understanding of the mechanisms underlying stress responses and supplies significant transcriptome resources for studying biological questions in non-model fish species.