Definitive haematopoiesis occurs during the lifetime of an individual, which continuously replenishes all blood and immune cells. During embryonic development, haematopoietic stem cell (HSC) formation is tightly controlled by growth factors, signalling molecules and transcription factors. But little is known about roles of the cytochrome P450 (CYP) 2 family member in the haematopoiesis. Here we report characterization and functional studies of Cyp2aa9, a novel zebrafish Cyp2 family member. And demonstrate that the cyp2aa9 is required for the HSC formation and homeostasis. Knockdown of cyp2aa9 by antisense morpholino oligos resulted the definitive HSC development is defective and the Wnt/β-catenin activity becomes reduced. The impaired HSC formation caused by cyp2aa9 morpholino can be rescued by administration of PGE2 through the cAMP/PKA pathway. Furthermore, the in vivo PGE2 level decreases in the cyp2aa9 morphants, and none of the PGE2 precursors is able to rescue phenotypes in the Cyp2aa9-deficient embryos. Taken together, these data indicate that Cyp2aa9 is functional in the step of PGE2 synthesis from PGH2, thus promoting Wnt activation and definitive HSC development.