HMGA2 protein belongs to the High Mobility Group A (HMGA) family of architectural transcription factors. These proteins establish a network of protein−protein and protein−DNA interactions resulting in the formation of enhanceosomes at promoters and enhancers regulating the expression of several genes. HMGA2 dysregulation, as a result of specific chromosomal rearrangements, has been identified in a variety of common benign mesenchymal tumors, and transgenic mice expressing a truncated form of HMGA2 protein demonstrated a causal relationship between the expression of the HMGA2 protein and tumorigenesis. In this paper, using several recombinant mutant proteins, we have investigated the role played by the different domains of HMGA2 in protein−protein and protein−DNA interaction. Using the IFN-β gene as a model, we have shown that a short region of HMGA2, comprising the second DNA-binding domain, is critical for enhancing the NF-κB complex formation, for binding to the PRDII element, and also for protein−protein interaction with the NF-κB p50 subunit. Moreover, we have analyzed the interaction of HMGA2 mutant proteins with different DNA targets demonstrating that the absence of the C-terminal tail alters HMGA2/DNA complexes in a subset of DNA sequences. Our results suggest possible implications for the role of HMGA2 in tumorigenesis.