The Hox family transcription factors control diversified morphogenesis during development and evolution. They function in concert with Pbc cofactor proteins. Pbc proteins bind the Hox hexapeptide (HX) motif and are thereby thought to confer DNA binding specificity. Here we report that mutation of the AbdA HX motif does not alter its binding site selection but does modify its transregulatory properties in a gene-specific manner in vivo. We also show that a short, evolutionarily conserved motif, PFER, in the homeodomain-HX linker region acts together with the HX to control an AbdA activation/repression switch. Our in vivo data thus reveal functions not previously anticipated from in vitro analyses for the hexapeptide motif in the regulation of Hox activity.