Acinetobacter baumannii is intrinsically resistant to fosfomycin; however, the mechanisms underlying this resistance are poorly understood.To identify and characterize genes that contribute to intrinsic fosfomycin resistance in A. baumannii.More than 9000 individual transposon mutants of the A. baumannii ATCC 17978 strain (fosfomycin MIC ≥1024 mg/L) were screened to identify mutations conferring increased susceptibility to fosfomycin. In-frame deletion mutants were constructed for the identified genes and their susceptibility to fosfomycin was characterized by MIC determination and growth in the presence of fosfomycin. The effects of these mutations on membrane permeability and peptidoglycan integrity were characterized. Susceptibilities to 21 antibiotics were determined for the mutant strains.Screening of the transposon library identified mutants in the ampD and anmK genes, both encoding enzymes of the peptidoglycan recycling pathway, that demonstrated increased susceptibility to fosfomycin. MIC values for in-frame deletion mutants were ≥42-fold (ampD) and ≥8-fold (anmK) lower than those for the parental strain, and growth of the mutant strains in the presence of 32 mg/L fosfomycin was significantly reduced. Neither mutation resulted in increased cell permeability; however, the ampD mutant demonstrated decreased peptidoglycan integrity. Susceptibility to 21 antibiotics was minimally affected by mutations in ampD and anmK.This study demonstrates that AmpD and AnmK of the peptidoglycan recycling pathway contribute to intrinsic fosfomycin resistance in A. baumannii, indicating that inhibitors of these enzymes could be used in combination with fosfomycin as a novel treatment approach for MDR A. baumannii.