Adult hematopoietic stem cells (HSCs) are routinely used to reconstitute hematopoiesis after myeloablation; however, transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number, or poor homing, engraftment, or self-renewal. Here we report that mouse and human HSCs express prostaglandin E₂ (PGE₂) receptors, and that short-term ex vivo exposure of HSCs to PGE₂ enhances their homing, survival, and proliferation, resulting in increased long-term repopulating cell (LTRC) and competitive repopulating unit (CRU) frequency. HSCs pulsed with PGE₂ are more competitive, as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon serial transplantation, with full multilineage reconstitution. PGE₂ increases HSC CXCR4 mRNA and surface expression, enhances their migration to SDF-1 in vitro and homing to bone marrow in vivo, and stimulates HSC entry into and progression through cell cycle. In addition, PGE₂ enhances HSC survival, associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. Our results define novel mechanisms of action whereby PGE₂ enhances HSC function and supports a strategy to use PGE₂ to facilitate hematopoietic transplantation.