The pituitary tumor-transforming gene-binding factor (PBF) is a relatively uncharacterised proto-oncogene, which is overexpressed in thyroid tumours. PBF elicits tumor growth in nude mice, whilst thyroid targeted transgenic overexpression in the PBF-Tg mouse induces hyperplasia and macrofollicular lesions, accompanied by induction of the E2 ubiquitin ligase Rad6. Our previous unpublished data showed that PBF binds to p53, and reduces stimulation of downstream target genes by competitive binding. Further, half-life studies of p53 showed reduced p53 stability when PBF was overexpressed in K1 and TPC-1 thyroid papillary cancer cell lines, and ubiquitination assays confirmed this was due to increased ubiquitination and subsequent degradation by the proteasome. Now, GST pull-down assays demonstrate direct binding between PBF and MDM2, the principal negative regulator of p53. The competitive inhibitor of p53-MDM2 binding, Nutlin-3, revealed that the increased degradation of p53 observed when PBF was overexpressed was mediated by MDM2. No change in p53-MDM2 binding stringency was detected when PBF expression was ameliorated by siRNA treatment, and MDM2 subcellular localisation was unchanged by PBF overexpression in K1 cells. However, co-immunoprecipitation assays (TPC and K1 thyroid cells) revealed that PBF specifically interacts with Rad6, which has previously been shown to regulate p53 ubiquitination. Further, PBF-Tg mice demonstrated significantly induced genetic instability at 6 weeks of age, as determined by FISSR-PCR. Thus we propose that aberrantly expressed PBF functionally inactivates p53 via a complex interplay between Rad6 and MDM2, thus promoting genetic instability and tumorigenesis in thyroid cells and leading to thyroid cancer.