Population-based studies have demonstrated an association of single nucleotide polymorphisms close to the thyroid transcription factor forkhead box E1 (FOXE1) gene with thyroid cancer. The dysregulation of forkhead proteins is increasingly recognized to play a role in the development and progression of cancer. The objective of the study was to seek to identify novel mutations in FOXE1 in papillary thyroid cancer (PTC) and to assess the effect of these mutations on protein expression and transcriptional function on FOXE1 responsive promoters. METHODS: The study was conducted at two tertiary referral hospitals. The coding region of FOXE1 was sequenced in tissue-derived DNA or RNA from 120 patients with PTC and 110 patients with multinodular goiter (MNG). In vitro studies were performed to examine the protein expression and transcriptional function of FOXE1 mutants. A molecular model of the forkhead domain (FHD) of FOXE1 was generated using the SWISS-MODEL online server with the three-dimensional structure of FOXD3 as a template. RESULTS: Three somatic missense mutations were detected in PTC resulting in the amino acid substitutions P54Q, K95Q, and L112F. One additional mutation was detected in a MNG (G140R). In vitro studies demonstrated marked impairment in transcriptional activation by all four FOXE1 mutants, which was not explained by differences in protein expression. Molecular modeling localized three of the mutations to highly conserved regions of the FHD. CONCLUSIONS: We have identified novel somatic mutations of FOXE1 in PTC. Mutational inactivation of FOXE1 is an uncommon event in thyroid tumors but may contribute to thyroid carcinogenesis and dedifferentiation in concert with other oncogenic drivers.