Drug resistance is the major setback of acute myeloid leukemia (AML) therapy. Notch proteins have demonstrated functional regulation in cell proliferation, differentiation, and apoptosis and thus may affect drug resistance. Our study aimed to identify the Notch-related gene profile in drug-resistant AML cells and provide potential strategies for resistant AML therapy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to detect the cytotoxicity of adriamycin toward K562 and drug-resistant K562/A02. Intracellular mean fluorescence intensity was monitored to reflect the intake of adriamycin by confocal microscopy. cDNA microarray was used to test the expression of 113 Notch signaling pathway-related genes in K562/A02 and K562. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot were used to validate the results from microarray. K562/A02 cells showed a 65-fold higher IC(50) to adriamycin and less intracellular accumulation of adriamycin than K562. cDNA microarray showed marked increases in binding of collagen and cell proliferation-related genes (CD44, DLL3, IL17B, NUMB, and NUMBL) and decreases in signal transduction and transcription factor activity related genes (FZD9, GBP2, GLI1, GLI2, IFNG, KRT5, Notch2, and Notch3). The change of gene expression was further validated by real-time RT-PCR and western blot. Notch signaling pathway-related genes may contribute to the drug resistance of AML.