Bacteria cell walls contain many repeating glycan structures, such as peptidoglycans, lipopolysaccharides, teichoic acids, and capsular polysaccharides. Their synthesis starts in the cytosol, and they are constructed from a glycan lipid carrier, undecaprenyl phosphate (C55P), which is essential for cell growth and survival. The lipid derivative undecaprenol (C55OH) is predominant in many Gram-positive bacteria but has not been detected in Gram-negative bacteria; its origin and role have thus remained unknown. Recently, a homologue of diacylglycerol kinase (DgkA) in Escherichia coli (E. coli) was demonstrated to be an undecaprenol kinase (UK) in the Gram-positive bacterium Streptococcus mutans (S. mutans). In this study, we found that S. mutans UK was not only an undecaprenol kinase but also a Mg-ADP-dependent undecaprenyl phosphate phosphatase (UpP), catalyzing the hydrolysis of C55P to C55OH and a free inorganic phosphate. Furthermore, the naturally undetectable C55OH was observed in E. coli cells expressing S. mutans dgkA, supporting the phosphatase activity of UK/UpP in vivo. These two activities were indispensable to each other and utilized identical essential residues binding to their substrates, suggesting that both activities share the same active site and might involve a direct phosphoryl transfer mechanism. This study revealed a unique membrane enzyme displaying bifunctional activities determined by substrate binding and C55OH production. The reciprocal conversion of C55P and the undecaprenol pool efficiently regulate cell wall synthesis, especially in Gram-positive bacteria.