To investigate the interacting protein partners of the global regulator YmdB from Bacillus subtilis, co-immunoprecipitation was performed using an N-terminal HA-tagged YmdB expressed from its native chromosomal locus under an endogenous promoter. The interactome complexes were analyzed by LC-MS/MS on an Orbitrap Fusion mass spectrometer. The interactome revealed proteins involved in gene expression, transcription, and translation, as well as metabolism, proton transmembrane transport, and several proteins of unknown function.