The syncytiotrophoblast develops as a result of the fusion of placental villous cytotrophoblast cells. These multinuclear cells are essential for fetal-maternal exchange and production of pregnancy-related hormones. Placental deficiency is considered a major risk factor associated with intrauterine growth restriction and preeclampsia. Better understanding of placental patho-physiology requires knowledge on gene expression regulation involved in trophoblast differentiation. Using the well-established in vitro trophoblast differentiation model, we have performed a microarray analysis on mRNA expression in trophoblast and syncytiotrophoblast cell cultures. Dramatic changes in gene expression patterns were detected during trophoblast differentiation. As many as 3524 novel and known genes are found to be up- or down regulated for more than 2-fold. Real-time PCR analysis of a group of genes confirmed the reliability of the microarray data. Overall, this study provided a global view on the molecular events underlying trophoblast differentiation. Subsequent characterization on regulation and function of the identified genes may lead to discovery of new pathways important for placental differentiation and function.