Hypoxia-inducible factor (HIF) is an important transcription factor under hypoxic condition in many organisms, and plays a key role in the induction of hypoxia tolerance. It is necessary to establish a hypoxia model for HIF and to perform further hypoxia tolerance research. To investigate the value of Daphnia as a model organism in hypoxia precondition, we developed a preconditioning protocol with a model organism, Daphnia pulex. We found that two episodes of exposure to hypoxic solution resulted in enhanced hypoxia tolerance which is dependent on HIF. Comparative genomic analysis was also made to highlight the homology of HIF-related genes among Daphnia, fruitfly and human. We found that Daphnia is suitable for the study of human hypoxic injury as a model organism.