Klebsiella pneumoniae, an opportunistic pathogen, is pervasively distributed across the world. Its escalating antibiotic resistance poses a serious threat to global public health. The mechanisms behind this resistance remain largely elusive. In this study, we performed antibiotic susceptibility testing on several clinical isolates of Klebsiella pneumoniae, and a reference strain ATCC13883, and then analysed their transcriptomic profiles to identify genes and pathways associated with antibiotic resistance. Our results showed that a clinical isolate DY16KPN may counteract antibiotics by enhancing the biosynthesis of building blocks of bacterial cell, such as fatty acids, proteins, and DNA, and reducing transmembrane transport. Increased butanoate metabolism and lipopolysaccharide biosynthesis may also contribute to the drug-resistance of Klebsiella pneumoniae. Additionally, we identified resistance-promoting mutations in gene promoter regions, which regulate the activity of downstream drug-resistant genes and pathways. Our results also demonstrated that DY16KPN counterbalances the trimethoprim/sulfamethoxazole-mediated inhibitory effect on the synthesis of tetrahydrofolates and DNA by up-regulating the expression of targeted enzymes of trimethoprim/sulfamethoxazole, dihydrofolate reductase and dihydropteroate synthase.