Fish routinely experience environmental hypoxia and have evolved various strategies to tolerate this challenge. Given the key role of the corticotropin-releasing factor (CRF) system in coordinating the response to stressors and its cardioprotective actions against ischemia in mammals, we sought to characterize the cardiac CRF system in zebrafish and its role in hypoxia tolerance. We established that all genes of the CRF system, the ligands CRFa, CRFb, urotensin 1 (UTS1) and urocortin 3 (UCN3), the two receptor subtypes (CRFR1 and CRFR2), and the binding protein (CRFBP) are expressed in the heart of zebrafish: crfr1 > crfr2 = crfbp > crfa > ucn3 > crfb > uts1. In vivo, exposure to 5% O2 saturation for 15 min and 90 min recovery resulted in 4-5 fold increases in whole heart crfb and ucn3 mRNA levels but did not affect the gene expression of other CRF system components. In vitro, as assessed by monitoring caspase 3 activity and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, pre-treatment of excised whole hearts with CRF or UCN3 for 30 min prevented the increase in apoptosis associated with exposure to 1% O2 saturation for 30 min and 24 h recovery. Lastly, addition of the non-selective CRF receptor antagonist, αh-CRF(9-41), prevented the cytoprotective effects of CRF. We show that the CRF system is expressed in fish heart, is up-regulated by hypoxia, and is cytoprotective. These findings identify a novel role for the CRF system in fish and a new strategy to tolerate hypoxia.