Poly-γ-glutamic acid (PGA) is a promising polymer that comprises d- and l-glutamic acid (Glu) connected via an amide bond. PGA is biosynthesized by a transmembrane enzyme complex composed of PgsB, PgsC, and PgsA. However, the detailed reaction, especially the mechanism for introducing d-Glu residues into PGA, remains elusive. We herein report isotope tracer experiments with deuterated l- and d-Glu and demonstrate that epimerization of a growing peptide is involved in PGA biosynthesis.