The high-mobility-group A2 protein (HMGA2) plays important functional roles in transcriptional regulation, DNA replication and chromatin structure. In this study, the effect of histone deacetylase inhibition on the transcriptional activity of the Hmga2 gene was investigated in vivo both at the endogenous gene level and in a variety of cell lines using transiently transfected promoter constructs. Trichostatin A (TSA) repressed both transfected murine and human Hmga2 promoter constructs 3-8-fold in NIH3T3, F9 and HeLa cells. Steady-state Hmga2 mRNA levels in NIH3T3 cells decreased 4-5-fold following TSA treatment, while pre- treatment of NIH3T3 cells with the transcriptional inhibitor, actinomycin D, completely blocked TSA mediated repression of the Hmga2 gene. Cross-linked chromatin immunoprecipitation (X-ChIP) analysis revealed a 5-6-fold decrease in endogenous Hmga2 promoter bound Sp1 and Sp3 proteins following TSA treatment in parallel with observed loss of acetylated histone H3 and H4. In addition, the poly-pyrimidine-tract-binding protein (PTB) was observed to bind to the Hmga2 promoter in both TSA treated and untreated NIH3T3 cells. Together, these results suggest TSA treatment leads to a decrease in Hmga2 gene transcription, and a significant decrease in promoter bound Sp1, Sp3 and acetylated histones H3 and H4.