Pituitary homeobox 2 (PITX2), a Paired-like homeodomain transcription factor and a downstream effector of β-catenin signaling, plays substantial roles in normal embryonic development but its possible involvement in tumorigenesis was unknown. In this study, we extend its function in human cancer. Remarkably, we found that PITX2 was frequently expressed in human follicular cell-derived (papillary, follicular and anaplastic) thyroid cancer tissues but not in normal thyroids, indicating for the first time that overactivated PITX2 may contribute to thyroid cancer. Cell-based and biochemical studies were performed to uncover the molecular mechanism of PITX2 action in thyroid cancer. Knockdown of PITX2 gene expression in human thyroid cancer cells significantly reduced cell proliferation and soft-agar colony formation. Biochemical analysis of cell cycle regulators upon PITX2 knockdown revealed downregulation of Cyclin D1, Cyclin D2 and dephosphorylation of Rb. Chromatin immunoprecipitation and promoter reporter assay indicated that Cyclin D2 was a direct target gene of PITX2. Consistently, we observed that high expression levels of Cyclin D2 were frequently associated with PITX2 expression in follicular cell-derived thyroid cancer tissues. to confirm our results in vivo, we took advantage of a mouse model of thyroid cancer (TRbeta(PV/PV) mouse). Consistently, the aberrant elevation of PITX2 levels in the thyroid cancer of TRbeta(PV/PV) mice was accompanied by upregulation of Cyclin D1, Cyclin D2 and increased phosphorylation of Rb. Collectively, our findings demonstrate that the overactivated PITX2-Cyclin D2 pathway promotes thyroid tumorigenesis, and they provide the first evidence implicating an oncogenic role of PITX2 in human cancer.