Propionibacterium freudenreichii (P. freudenreichii) has various biotechnological applications, notably in the ripening of Swiss-type cheese, where it utilizes the two enantiomers of lactate as the main carbon source, contributing to flavor development and eye formation. Here, we genotypically characterized two P. freudenreichii strains unable to catabolize L-lactate through whole-genome sequencing and variant calling, using P. freudenreichii FAM-14222 as the reference genome, which highlighted a mutation in the lutB gene in both strains. This gene is part of the lutABC operon, which has been previously linked to lactate utilization in other bacterial species. Subsequently, we successfully restored the strains’ ability to utilize L-lactate by following an adaptive laboratory evolution approach, which involved repeated subculturing in a medium containing L-lactate as the main carbon source. Sequencing of the lutB gene confirmed that isolates with a restored ability to utilize L-lactate had also reverted the mutation back to wild-type, supporting the involvement of the lutABC operon in L-lactate catabolism in P. freudenreichii. Moreover, the phenotype of the two L-lactate-negative strains was confirmed under cheesemaking conditions, highlighting the potential of the strains as cheese ripening cultures. Lactate catabolism is of paramount importance in Propionibacterium freudenreichii, particularly for its industrial applications, such as Swiss-type cheese ripening. Nevertheless, the genetic background of this metabolic process is not fully understood. In our study, we developed an adaptive laboratory evolution-based approach for the elucidation of L-lactate catabolism, starting from two strains unable to utilize L-lactate. Our results delivered experimental evidence of the role of the lutABC operon in this process, as opposed to the widespread theory of L-lactate dehydrogenasemediated oxidation. A deeper understanding of this metabolic pathway will be beneficial for a more efficient selection of industrial strains, as well as for metabolic engineering.