AbstractLysophosphatidic acid (LPA), an extracellular lipid mediator, exerts multiple bioactivities through activating G protein-coupled receptors. LPA receptor 3 (LPA3) is a member of the endothelial differentiation gene family, which regulates differentiation and development of the circulation system. However, the relationship among the LPA receptors (LPARs) and erythropoiesis is still not clear. In this study, we found that erythroblasts expressed both LPA1 and LPA3, and erythropoietic defects were observed in zLPA3 antisense morpholino oligonucleotide-injected zebrafish embryos. In human model, our results showed that LPA enhanced the erythropoiesis in the cord blood-derived human hematopoietic stem cells (hHSCs) with erythropoietin (EPO) addition in the plasma-free culture. When hHSCs were treated with Ki16425, an antagonist of LPA1 and LPA3, erythropoietic process of hHSCs was also blocked, as detected by mRNA and protein expressions of CD71 and GlyA. In the knockdown study, we further demonstrated that specific knockdown of LPA3, not LPA1, blocked the erythropoiesis. The translocation of β-catenin into the nucleus, a downstream response of LPAR activation, was blocked by Ki16425 treatment. In addition, upregulation of erythropoiesis by LPA was also blocked by quercetin, an inhibitor of the β-catenin/T-cell factor pathway. Furthermore, the enhancement of LPA on erythropoiesis was diminished by blocking c-Jun-activated kinase/signal transducer and activator of transcription and phosphatidylinositol 3-kinase/AKT activation, the downstream signaling pathways of EPO receptor, suggested that LPA might play a synergistic role with EPO to regulate erythropoietic process. In conclusion, we first reported that LPA participates in EPO-dependent erythropoiesis through activating LPA3.