The combination of polymyxins and chloramphenicol possesses synergistic killing activity against New Delhi metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae. This systems study examined the transcriptomic responses to the polymyxin/chloramphenicol combination in clinical NDM-producing K. pneumoniae isolate S01. Klebsiella pneumoniae S01 (initial inoculum ~108 CFU/mL) was treated with polymyxin B (1 mg/L, continuous infusion) or chloramphenicol [maximum concentration (Cmax) = 8 mg/L, half-life (t1/2) = 4 h], alone or in combination, using an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model to mimic their pharmacokinetics in patients. Transcriptomic profiles of bacterial samples collected at 0, 0.25, 1, 4 and 24 h were examined using RNA sequencing (RNA-Seq). Chloramphenicol monotherapy significantly increased the expression of genes involved in ribosomal synthesis across the entire 24-h treatment, reflective of chloramphenicol-mediated inhibition of protein synthesis. The effect of polymyxin B was rapid and no major pathways were perturbed at later time points (4 h and 24 h). Combination treatment yielded the highest number of differentially expressed genes, including a large number observed following chloramphenicol monotherapy, in particular carbohydrate, nucleotide, amino acid and cell wall metabolism. Notably, chloramphenicol alone and in combination with polymyxin B significantly inhibited the expression of the arn operon that is responsible for lipid A modification and polymyxin resistance. These results indicate that the polymyxin/chloramphenicol combination displayed persistent transcriptomic responses over 24 h mainly on cell envelope synthesis and metabolism of carbohydrates, nucleotides and amino acids.