Prostaglandin (PG) E₂ may regulate invasiveness of human placenta because we have previously reported stimulation of migration of placental trophoblasts by PGE₂ acting through EP1 receptor and activating calpain. RhoA GTPase and its important effector Rho kinase (ROCK) have also been previously shown to regulate trophoblast migration. Using immortalized HTR-8/SVneo trophoblast cells and first trimester human chorionic villus explant cultures on matrigel we further examined the role of RhoA/ROCK and MAP kinase (ERK1/2) pathways on PGE₂-mediated stimulation of trophoblast migration. Migration of cytotrophoblasts was shown to be inhibited by treatment of the trophoblast cell line and chorionic villus explants with either cell permeable C3 transferase or selective RhoA siRNA. These inhibitions were significantly mitigated by addition of PGE₂, an EP1/EP3 agonist or an EP3/EP4 agonist, suggesting that RhoA plays an important role in trophoblast migration but may not be obligatory for PGE₂ action. Treatment of HTR-8/SVneo cells with non-selective Rho kinase (ROCK) inhibitor Y27632 or ROCK siRNAs inhibited migration of these cells, which could not be rescued with PGE₂ or the other two EP-agonists suggesting the obligatory role of ROCK in PGE₂-induced migratory response. Furthermore, MEKK inhibitor U0126 abrogated PGE₂-induced migration of trophoblasts and PGE₂ or the other two EP-agonists stimulated ERK1/2 activation in trophoblasts, which was not abrogated by pretreatment with C3 transferase, indicating that ERK signaling pathway is an efficient alternate pathway for RhoA in PGE₂-mediated migration of trophoblasts. These results suggest that ROCK and ERK1/2 play more important roles than RhoA in PGE₂-mediated migration stimulation of first trimester trophoblasts.