Pituitary tumor-transforming gene binding factor (PBF) is a ubiquitous glycoprotein which is over-expressed in thyroid, breast and other endocrine cancers, and modulates cellular invasion, radioiodine uptake and thyroid hormone efflux. Papillary thyroid cancer patients with high PBF expression show decreased disease-specific survival compared to those with lower expression. PBF expression has recently been correlated with breast cancer metastasis and colon cancer extra-mural vascular invasion. Previously classified as a proto-oncogene, the first ten substitution-missense mutations of PBF have recently been reported via the COSMIC and TCGA databases, suggesting PBF may in fact be an oncogene. We have therefore examined the biological implications of all 10 mutations in thyroid and breast cells. Substitution mutations generally resulted in clear alterations in the 3D structure of PBF. Western blotting revealed that mutations C51R and G106R inhibited PBF dimerisation and glycosylation in vitro. Anisomycin half life studies in SW1736 thyroid and MCF7 breast cancer cells demonstrated that the C51R mutation resulted in increased protein stability compared to wild type, whereas V55I, W59F, R87C and S103L, G106R, G106V, G106W and R146W mutants were less stable. BrdU proliferation assays revealed that C51R and V55I induced a significant proliferative advantage in thyroid and breast cells compared with WT, whilst R140W significantly repressed proliferation. Mutant C51R was mainly confined to the endoplasmic reticulum while R140W was apparent in the Golgi apparatus. Cell invasion assays demonstrated significantly reduced cell invasion in mutant C51R, accompanied by increased binding to the cortical actin binding protein cortactin. Both C51R and R140W showed decreased cellular migration, but retained the ability to repress radioiodide, a functional hallmark of WT PBF. This is the first description of mutations in PBF, a gene implicated in the initiation and progression of thyroid and other cancers. Taken together our data suggest that mutations in PBF might represent rare novel aetiological events in human cancer.