This is an open-access article distributed under the terms of the Creative Commons Attribution License. ; Extradenticle (Exd) and Homothorax (Hth) function as positive transcriptional cofactors of Hox proteins, helping them to bind specifically their direct targets. The posterior Hox protein Abdominal-B (Abd-B) does not require Exd/Hth to bind DNA; and, during embryogenesis, Abd-B represses hth and exd transcription. Here we show that this repression is necessary for Abd-B function, as maintained Exd/Hth expression results in transformations similar to those observed in loss-of-function Abd-B mutants. We characterize the cis regulatory module directly regulated by Abd-B in the empty spiracles gene and show that the Exd/Hth complex interferes with Abd-B binding to this enhancer. Our results suggest that this novel Exd/Hth function does not require the complex to bind DNA and may be mediated by direct Exd/Hth binding to the Abd-B homeodomain. Thus, in some instances, the main positive cofactor complex for anterior Hox proteins can act as a negative factor for the posterior Hox protein Abd-B. This antagonistic interaction uncovers an alternative way in which MEIS and PBC cofactors can modulate Abd-B like posterior Hox genes during development. ; This work was supported by the Programa Consolider, MICINN, European Regional Development Fund (FEDER), and Junta de Andalucía to JC-GH and by an EMBO short-term fellowship to MLR. YG was supported by the CNRS, Université de la Méditerranée, and grants from CEFIPRA, ANR, FRM, and ARC, and by a fellowship from MRT CEFIPRA to NS. ; Peer reviewed