The rapid increase of polymyxin-resistant MDR K. pneumoniae isolates has become a major global concern. This study aimed to investigate lipid A profiles and metabolomes of PBS and PBR MDR K. pneumoniae. Three paired of clinical isolates (Kp AHT 8 [PB MIC 64mg/L] vs AHT 7 [0.25mg/L], Kp AHT 16 [32mg/L] vs AHT 15 [0.5mg/L], and Kp AHT 18 [64mg/L] vs AHT 17 [1mg/L] were examined using liquid chromatography mass spectrometry and differences in the lipid A profiles and metabolomes were identified through multivariate and univariate statistics. The predominate forms of lipid A in these paired isolates were hexa-acylated, and lipid A species from PBR isolates were mostly modified with 4-amino-4-deoxy-L-arabinose. The metabolomic profiles of PBS and PBR isolates were significantly different. In detail, UDP-N-acetyl-D-glucosamine, UDP-N-acetyl-D-mannosaminuronate associated with lipid A biosynthesis were significantly depleted in all PBR isolates (>1.5 FC, p < 0.05), compared to their parent isolates. The intermediate metabolites in carbohydrate metabolic pathway, particularly pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle, were significantly lower in all PBR isolates (approximately >1.5 FC, p < 0.05). Most nucleotide metabolites were also lower in abundance in PBR isolates. Two important metabolites associated with the glycerophospholipid (GPL) metabolism, sn-glycerol-3-phosphate and sn-glycero-3-phosphoethanolamine were significantly lower in abundance in all PBR isolates (> 2 FC, p < 0.05). Remarkably lower GPL levels were evident in PBR AHT 16 and AHT 18 (approximately >1.5 FC), compared to their parent isolates, while no significant GLP changes were observed in PBR Kp AHT 8. This study is the first to reveal that polymyxin resistance causes different metabolism in PBR MDR K. pneumoniae and provided valuable mechanistic information for the development of rational polymyxin combinations against PBRK. pneumoniae.