Two clonally identical L. monocytogenes strains, N843-10 and N843-15 isolated five years apart from the same persistent human prosthetic hip joint infection (PJI) were compared. In comparison to the parent strain N843-10, strain N843-15 isolated five years later showed atypical growth and aggregation behavior, as well as severe morphological defects including filamentation. In further phenotypic analysis this strain showed increased antibiotic sensitivity, diminished carbon source utilization, stress tolerance and biofilm production compared to N843-10. Furthermore, both strains were significantly diminished in virulence when compared to other L. monocytogenes strains. Genomic analysis uncovered genetic mutations in N843-15 causing loss of function of PrfA, RNAse J1 and HtrA proteins to explain the major phenotypic alterations observed in this strain. Overall our findings indicate that L. monocytogenes N843 strain lost functions that are dispensable for its survival in the joint environment to enable long-term PJI persistence with minimal detrimental effects to the host.