FoxA1-3 (formerly HNF3alpha, -beta, and -gamma), members of the FoxA subfamily of forkhead transcription factors, function as initial chromatin-binding and chromatin-remodeling factors in a variety of tissues, including liver and pancreas. Despite essential roles in development and metabolism, regulation of FoxA factors is not well understood. This study examines a potential role for acetylation in the regulation of FoxA chromatin binding and remodeling. Using in silico analysis, we have identified 11 putative p300 acetylation sites within FoxA1, five of which are located within wings 1 and 2 of its winged-helix DNA-binding domain. These polypeptide structures stabilize FoxA DNA and chromatin binding, and we have demonstrated that acetylation attenuates FoxA binding to DNA and diminishes its ability to remodel chromatin. FoxA acetylation is inhibited by chromatin binding. We propose a model whereby stable chromatin binding protects the FoxA DNA-binding domain from acetylation to preserve chromatin binding and remodeling by FoxA factors in the absence of extracellular cues.