PCIF1 is a TRAF and POZ domain containing nuclear factor that interacts with and inhibits transactivation of pancreatic homeodomain transcription factor PDX-1. Here, we demonstrate interaction of endogenous PDX-1 and PCIF1 in MIN6 insulinoma cells. Within PCIF1, the TRAF and POZ domains are both required for physical and functional interaction with the C-terminus of PDX-1, whereas the C-terminal domain of PCIF1 directs its nuclear localization. A human PDX-1 mutation associated with diabetes, E224K, disrupts the ability of PCIF1 to inhibit PDX-1 transactivation, suggesting that the interaction between PDX-1 and PCIF1 is required for normal glucose homeostasis. Inhibition of transactivation occurs by a mechanism distinct from the classical role of POZ domains to recruit co-repressors and histone deacetylases. Understanding the functional roles of PCIF1 domains may have application to therapeutic β-cell replacement strategies involving PDX-1 for the treatment of diabetes.