Objective Imatinib mesylate (IM) is a tyrosine kinase inhibitor selective for BCR-ABL and indicated for the treatment of chronic myeloid leukemia. It has recently been demonstrated that IM also targets other cellular components. Considering the significant role of telomerase in malignant transformation, we studied the effect of IM on telomerase activity (TA) and regulation in BCR-ABL–positive and –negative cells, sensitive and resistant to IM. Materials and Methods Through combining telomeric repeat amplification protocol for detecting TA, reverse transcription polymerase chain reaction and Western blots for detecting RNA and protein levels of telomerase regulating proteins and fluorescence-activated cell sorting analysis, we showed that IM targets telomerase and the signal transduction cascade upstream of it. Results IM significantly inhibited TA in BCR-ABL–positive and –negative cells and in chronic myeloid leukemia patients. TA inhibition was also observed in BCR-ABL positive cells resistant to IM at drug concentrations that did not lead to a reduction in BCR-ABL expression. In addition, a reduction in phosphorylated AKT and phosphorylated PDK-1 was also detected following IM incubation. Conclusions We demonstrate an inhibitory effect of IM on TA and on the AKT/PDK pathway. Because this effect was observed in cell expressing the BCR-ABL protein as well as cells not expressing it, and in cells sensitive as well as resistant to IM, it is reasonable to assume that the inhibitory effect of IM on TA is not mediated through known IM targets. The results of this study show that cells resistant to IM with regard to its effect on BCR-ABL could still be sensitive to IM treatment regarding other cellular components.