Objective: To understand whether verapamil (VER) resistance development in the multidrug-resistant cell line and its mechanism. Methods: K562/ADM/VER cell subline resistant to verapamil was established through a gradual increase of VER concentration in the media. MTT method was used to assay resistance to VER, cross resistance to dipyriamole (DPM), cyclosporin A (CsA) in the cells, and HPLC and spectrofluorometer to detect intracellular accumulation of VER or ADM respectively, as well as S-P immunocytochemical technique for detection of genes expression. Results: It were observed that 7.9—fold increase in VER resistance, significantly reduced intracellular accumulation of VER or ADM and also develop across resistance to DPM and CsA in K562/ADM/VER cells, compared with its parent cell, K562/ADM. High-level of p-glycoprotein(pgp), middle-level of p53, p16, was present in two cell lines without expression of GSTPI, C-myc, C-myc, C-fos and C-erbB-2. Bc1–2 protein expression was found only in K562/ADM cells. Conclusion: K562/ADM cells were capable of being induced to develop resistance to VER.