Bacteria synthesize numerous sphingolipids with various physiological functions. Despite their roles in mediating host inflammation, cellular differentiation, and protection from environmental stress, their biosynthetic pathway remained undefined since several essential eukaryotic ceramide synthesis enzymes have no obvious bacterial homologue. Using genetic and biochemical approaches, we identified the enzymes required for bacterial ceramide synthesis. Bioinformatic analyses revealed the presence of these genes in a broad range of bacterial taxa and led to our discovery of the first Gram-positive species to produce ceramide. Biochemical experiments with purified proteins support a model in which the bacterial pathway operates in a different order than in eukaryotes. Furthermore, phylogenetic analyses are consistent with the independent evolution of the bacterial and eukaryotic ceramide synthesis pathways.