Lanthipeptides are natural products that belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). They contain characteristic lanthionine (Lan) or methyllanthionine (MeLan) structures that contribute to their diverse biological activities. Despite its structurally diverse set of 30 substrates, the highly substrate-tolerant lanthipeptide synthetase ProcM is shown to display high selectivity for formation of a single product from selected substrates. Mutation of the active site zinc ligands to alanine or the unique zinc ligand Cys971 to histidine resulted in a decrease of the cyclization rate, especially for the second cyclization of the substrates ProcA1.1, ProcA2.8, and ProcA3.3. Surprisingly, for ProcA3.3 these mutations also altered the regioselectivity of cyclization resulting in a new major product. ProcM was not able to correct the ring topology of incorrectly cyclized intermediates and products, suggesting that thermodynamic control is not operational. Collectively, the data in this study suggest that the high regioselectivity of product formation is governed by the selectivity of the initially formed ring.