Abstract: In order to understand the molecular mechanism of rafoxanide（RAF） enhancing the antibacterial activity of colistin（COL） against Klebsiella pneumoniae, the minimum inhibitory concentration（MIC） and the partial inhibitory concentration（FIC） index of RAF and COL alone or combined against Klebsiella pneumoniae were determined by microbroth dilution method. Then, transcriptomic methods were used to analyze the clustering heat map of drug resistance genes, differential expression gene volcano map, KEGG pathway enrichment, KEGG functional annotation, and differential gene expression of small molecule metabolism and transport after the combination of RAF and COL for 4 h. The results showed that the MIC value of COL against KP1 was 128 μg/mL, which could be reduced to 0.25 μg/mL when combined with RAF. RAF and COL had a synergistic effect with 0.0078 ≤FIC≤0.008 8. Compared with COL alone, the expression of resistant genes bla_CTX-M, bla_SHV, aac（3）-Ⅱd, aph3-Ⅰ, aac（6′）-Ⅰb, gyrA, gyrB, and parC decreased after combination therapy. A total of 2 017 differentially expressed genes were downregulated. The pathways of significant gene enrichment and gene number in down-regulated genes were aminoacyl-TRNA synthesis, porphyrin and chlorophyll metabolism, lipopolysaccharide biosynthesis, oxidative phosphorylation and ribosome. The pathways of significant enrichment mainly include carbohydrate metabolism, cofactor and vitamin metabolism, membrane transport, amino acid metabolism, energy metabolism and signal transduction. The differentially expressed genes related to small molecule metabolism and transport, such as glycerol metabolism, ribosome transport, oxidative phosphorylation, tricarboxylic acid cycle and antioxidant, were down-regulated. This indicated that RAF might enhance the antibacterial activity of COL by affecting the metabolism and transport functions of Klebsiella pneumoniae and related small molecules.