Although bacteria have diverse membrane proteins, the function of many of them remains unknown or uncertain even in Escherichia coli. In this study, to investigate the function of hypothetical membrane proteins, genome-wide analysis of phenotypes of hypothetical membrane proteins was performed under various envelope stresses. Several genes responsible for adaptation to envelope stresses were identified. Among them, deletion of YhcB, a conserved inner membrane protein of unknown function, caused high sensitivities to various envelope stresses and increased membrane permeability, and caused growth defect under normal growth conditions. Furthermore, yhcB deletion resulted in morphological aberration, such as branched shape, and cell division defects, such as filamentous growth and the generation of chromosome- less cells. The analysis of antibiotic susceptibility showed that the yhcB mutant was highly susceptible to various anti-folate antibiotics. Notably, all phenotypes of the yhcB mutant were completely or significantly restored by YhcB without the transmembrane domain, indicating that the localization of YhcB on the inner membrane is dispensable for its function. Taken together, our results demonstrate that YhcB is involved in cell morphology and cell division in a membrane localization-independent manner.