Cobamides are cobalt-containing cyclic tetrapyrroles involved in the metabolism of organisms from all domains of life but produced de novo only by some bacteria and archaea. The pathway is thought to involve up to 30 enzymes, five of which comprise the so-called "late" steps of cobamide biosynthesis. Two of these reactions activate the corrin ring, one activates the nucleobase, a fourth one condenses activated precursors, and a phosphatase yields the final product of the pathway. The penultimate step is catalyzed by a polytopic integral membrane protein, namely, the cobamide (5'-phosphate) synthase, also known as cobamide synthase. At present, the reason for the association of all putative and bona fide cobamide synthases to cell membranes is unclear and intriguing. Here, we show that, in Escherichia coli, elevated levels of cobamide synthase kill the cell by dissipating the proton motive force and compromising membrane stability. We also show that overproduction of the phosphatase that catalyzes the last step of the pathway or phage shock protein A prevents cell death when the gene encoding cobamide synthase is overexpressed. We propose that in E. coli, and probably all cobamide producers, cobamide synthase anchors a multienzyme complex responsible for the assembly of vitamin B12 and other cobamides. IMPORTANCE E. coli is the best-studied prokaryote, and some strains of this bacterium are human pathogens. We show that when the level of the enzyme that catalyzes the penultimate step of vitamin B12 biosynthesis is elevated, the viability of E. coli decreases. These findings are of broad significance because the enzyme alluded to is an integral membrane protein in all cobamide-producing bacteria, many of which are human pathogens. Our results may provide new avenues for the development of antimicrobials, because none of the enzymes involved in vitamin B12 biosynthesis are present in mammalian cells.