The effective treatment of carbapenemase-producing Klebsiella pneumoniae infection has been limited and required novel potential agents. Due to the novel drug development crisis, using old antimicrobial agents and combination therapy have been highlighted. This study focused on fosfomycin which inhibits cell wall synthesis and has potential activity on Enterobacteriaceae. We evaluated fosfomycin activity against carbapenemase-producing K. pneumoniae and characterized fosfomycin resistance mechanisms. Fosfomycin revealed effective activity against only 31.8% of carbapenemase-producing K. pneumoniae isolates. The major resistance mechanism was FosA3 production. The co-occurrence of FosA3 overexpression with the mutation of glpT (or loss of glpT) and/or uhpT was mediated high-level resistance (MIC>256 mg/L) to fosfomycin. Moreover, fosA3 silenced in sixteen fosfomycin-susceptible isolates and the plasmid carrying fosA3 of these isolates increased 32- to 64-fold of fosfomycin MICs in Escherichia coli DH5α transformants. The in vitro activity of fosfomycin combination with amikacin by checkerboard assay showed synergism and no interaction in six (16.2%) and sixteen isolates (43.3%), respectively. No antagonism of fosfomycin and amikacin was observed. Notably, the silence of aac (6)'-Ib and aphA6 was observed in amikacin-susceptible isolates. Our study suggests that the combination of fosfomycin and amikacin may be insufficient for the treatment of carbapenemase-producing K. pneumoniae isolates.