Background Hox proteins finely coordinate antero-posterior axis during embryonic development and through their action specific target genes are expressed at the right time and space to determine the embryo body plan. As master transcriptional regulators, Hox proteins recognize DNA through the homeodomain (HD) and interact with a multitude of proteins, including general transcription factors and other cofactors. HD binding specificity increases by protein–protein interactions with a diversity of cofactors that outline the Hox interactome and determine the transcriptional landscape of the selected target genes. All these interactions clearly demonstrate Hox-driven transcriptional regulation, but its precise mechanism remains to be elucidated. Results Here we report Antennapedia (Antp) Hox protein–protein interaction with the TATA-binding protein (TBP) and the formation of novel trimeric complexes with TFIIEβ and Extradenticle (Exd), as well as its participation in transcriptional regulation. Using Bimolecular Fluorescence Complementation (BiFC), we detected the interaction of Antp-TBP and, in combination with Förster Resonance Energy Transfer (BiFC-FRET), the formation of the trimeric complex with TFIIEβ and Exd in living cells. Mutational analysis showed that Antp interacts with TBP through their N-terminal polyglutamine-stretches. The trimeric complexes of Antp-TBP with TFIIEβ and Exd were validated using different Antp mutations to disrupt the trimeric complexes. Interestingly, the trimeric complex Antp-TBP-TFIIEβ significantly increased the transcriptional activity of Antp, whereas Exd diminished its transactivation. Conclusions Our findings provide important insights into the Antp interactome with the direct interaction of Antp with TBP and the two new trimeric complexes with TFIIEβ and Exd. These novel interactions open the possibility to analyze promoter function and gene expression to measure transcription factor binding dynamics at target sites throughout the genome.