Lysophosphatidic acid (LPA), a growth factor-like phospholipid, regulates numerous physiological functions, including cell proliferation and differentiation. In a previous study, we have demonstrated that LPA activates erythropoiesis by activating the LPA 3 receptor subtype (LPA3) under erythropoietin (EPO) induction. In the present study, we applied a pharmacological approach to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. In K562 human erythroleukemia cells, knockdown of LPA2 enhanced erythropoiesis, whereas knockdown of LPA3 inhibited RBC differentiation. In CD34(+) human hematopoietic stem cells (hHSC) and K526 cells, the LPA3 agonist 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted erythropoiesis, whereas the LPA2 agonist dodecyl monophosphate (DMP) and the nonlipid specific agonist GRI977143 (GRI) suppressed this process. In zebrafish embryos, hemoglobin expression was significantly increased by 2S-OMPT treatment but was inhibited by GRI. Furthermore, GRI treatment decreased, whereas 2S-OMPT treatment increased RBC counts and amount of hemoglobin level in adult BALB/c mice. These results indicate that LPA2 and LPA3 play opposing roles during RBC differentiation. The pharmacological activation of LPA receptor subtypes represent a novel strategies for augmenting or inhibiting erythropoiesis.