YhcB, an uncharacterized protein conserved across gamma-proteobacteria, is composed predominantly of a single Domain of Unknown Function (DUF 1043) with an N-terminal transmembrane -helix. Here, we show that E. coli YhcB is a conditionally essential protein that interacts with the proteins of the cell divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). We found 7 interactions of YhcB that are conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we identified several point mutations that abolished interactions of YhcB with FtsI and RodZ. The yhcB knock-out strain does not grow at 45{degrees}C and is hypersensitive to cell-wall acting antibiotics even in stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septa development. The 2.8 [A] crystal structure for the cytosolic domain from Haemophilus ducreyi YhcB shows a unique tetrameric -helical coiled-coil structure that combines parallel and anti-parallel coiled-coil intersubunit interactions. This structure is likely to organize interprotein oligomeric interactions on the inner surface of the cytoplasmic membrane, possibly involved in regulation of cell division and/or envelope biogenesis/integrity in proteobacteria. In summary, YhcB is a conserved and conditionally essential protein that is predicted to play a role in cell division and consequently or in addition affects envelope biogenesis. ImportanceOnly 0.8 % of the protein annotations in the UniProt are based on experimental evidence and thus, functional characterization of unknown proteins remains a rate-limiting step in molecular biology. Herein, the functional properties of YhcB (DUF1043) were investigated using an integrated approach combining X-ray crystallography with genetics and molecular biology. YhcB is a conserved protein that appears to be needed for the transition from exponential to stationary growth and is involved in cell division and/or envelope biogenesis/integrity. This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.