Invasion of cytotrophoblasts (CTBs) into uterine tissues is essential for placental development. To identify molecules regulating trophoblast invasion, mRNA signatures of purified villous (CTB, poor invasiveness) and extravillous trophoblasts (EVTs) (high invasiveness) isolated from first trimester human placentae and villous explant cultures, respectively, were compared using GeneChip analyses yielding 991 invasion/migration-related transcripts. Several genes involved in physiological and pathological cell invasion, including A disintegrin and metalloprotease-12, -19, -28, as well as Spondin-2, were up-regulated in EVTs. Pathway prediction analyses identified several functional modules associated with either the invasive or noninvasive trophoblast phenotype. One of the genes that was down-regulated in the invasive mRNA pool, heme oxygenase-1 (HO-1), was selected for functional analyses. Real-time PCR analyses, Western blotting, and immunofluorescence of first trimester placentae and differentiating villous explant cultures demonstrated down-regulation of HO-1 in invasive EVTs as compared with CTBs. Modulation of HO-1 expression in loss-of as well as gain-of function cell models (BeWo and HTR8/SVneo, respectively) demonstrated an inverse relationship of HO-1 expression with trophoblast migration in transwell and wound healing assays. Importantly, HO-1 expression led to an increase in protein levels and activity of the nuclear hormone receptor peroxisome proliferator activated receptor (PPAR) gamma. Pharmacological inhibition of PPARgamma abrogated the inhibitory effects of HO-1 on trophoblast migration. Collectively, our results demonstrate that gene expression profiling of EVTs and CTBs can be used to unravel novel regulators of cell invasion. Accordingly, we identify HO-1 as a negative regulator of trophoblast motility acting via up-regulation of PPARgamma.