Polymyxins are the last resort against multidrug-resistant Gram-negative bacteria; however, polymyxin resistance is emerging, including in a major nosocomial pathogen Klebsiella pneumoniae. The common resistance mechanisms involve lipid A modifications with phosphoethanolamine(pEtN) and 4-amino-4-deoxy-L-arabinose(L-Ara4N) mediated by chromosomal eptA and arnT, respectively. The recent discovery of plasmid-encoded pEtN transferase gene, mcr, indicates potential rapid horizontal dissemination of polymyxin resistance. This study aimed to investigate the interplay between pEtN and L-Ara4N transferase genes, mcr-1, eptA and arnT, in conferring polymyxin resistance in K. pneumoniae. Clinical K. pneumoniae II-503 carrying mcr-1 on a native plasmid was used to construct markerless deletion mutants. Single, double and triple deletions of mcr-1, eptA and arnT mutants were generated using the FLP/FRT recombination system. Polymyxin susceptibility was determined using MICs and static time-kill. Lipid A profiling was examined using LC-MS. All the mcr-1-carrying strains were resistant to polymyxin B with MICs of 8 mg/L, and static time-kills with polymyxin B at 8 mg/L were ineffective, indicating that the mcr-1 was the predominant polymyxin resistance mechanism. Reduced MICs of 0.5mg/L was observed for mutants with mcr-1 deleted. The static time-kills revealed significant killing of all mcr-1-deletion mutants with >3-log10cfu/mL reduction in bacterial viability at 1h following treatment. However, by 16h all these mutants demonstrated regrowth to the level similar to the control (~9 log10cfu/mL), with the exception of II-503Δmcr-1ΔeptAΔarnT which regrew to only 5.7±0.7 log10cfu/mL. Following regrowth, L-Ara4N-modified lipid A species were present in mutants that retained arnT but lacked mcr-1 (II-503Δmcr-1 and II-503Δmcr-1ΔeptA), while no modified lipid A species were observed in II-503Δmcr-1ΔarnT and II-503Δmcr-1ΔeptAΔarnT. In conclusion, mcr-1 and arnT, but not eptA, have major roles in conferring resistance towards polymyxin in K. pneumoniae II-503. The regrowth of the mutants lacking of both mcr-1 and arnT could due to other resistance mechanisms, which is currently under investigation using lipidomics and transcriptomics.