Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C-5 dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive geranyltransferase PirF shows that the enzyme utilizes a C-10 donor, with no C-5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C-5 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C-5 to a C-10 prenyltransferase, and vice versa. Lastly, we demonstrate that these enzymes may be used for the chemospecific attachment of C-5 or C-10 lipid groups on lanthipeptides, an unrelated class of RiPP natural products. These studies represent a rare example where prenyl donor specificity can be discretely altered, which expands the arsenal of synthetic biology tools for tuning biological activities of peptide natural products.