Vibrio cholerae annually infects millions worldwide, causing intense and life-threatening diarrheal disease. The pentameric cholera toxin subunit B (CtxB) binds to the ganglioside GM1 on epithelial cells, initiating endocytosis and downstream toxicity. We present the total chemical protein synthesis of L-CtxB and mirror-image D-CtxB using a three-segment N-to-C native chemical ligation approach. Oxidative folding produced the desired 62 kDa pentameric proteins, with L-CtxB capable of bind