The lantibiotic lacticin 3147 has been the focus of much research due to its broad spectrum of activity against many microbial targets, including drug-resistant pathogens. In order to protect itself, a lacticin 3147 producer must possess a cognate immunity mechanism. Lacticin 3147 immunity is provided by an ABC transporter, LtnFE, and a dedicated immunity protein, LtnI, both of which are capable of independently providing a degree of protection. In the study described here, we carried out an in-depth investigation of LtnI structure-function relationships through the creation of a series of fusion proteins and LtnI determinants that have been the subject of random and site-directed mutagenesis. We establish that LtnI is a transmembrane protein that contains a number of individual residues and regions, such as those between amino acids 20 and 27 and amino acids 76 and 83, which are essential for LtnI function. Finally, as a consequence of the screening of a bank of 28,000 strains producing different LtnI derivatives, we identified one variant (LtnI I81V) that provides enhanced protection. To our knowledge, this is the first report of a lantibiotic immunity protein with enhanced functionality.