Fentanyl is an opioid analgesic that it is widely used in cancer patients. Since there have been reports of effects of analgesic medications on the recurrence and development of resistance to treatment, influences of of fentanyl on MCF-7 and HEK293 cells were evaluated. Cell viability and apoptosis were assessed by MTT assay and flow cytometry, respectively. Gene expression analysis was performed by quantitative real-time PCR assay for the Oct4, Sox2 and Nanog genes as stem cell markers and Bax, Bcl2, and p53 genes as apoptosis markers. MTT assay results showed that fentanyl significantly inhibited the growth of MCF-7 cells in a dose-and time-dependent manner while significantly increasing apoptosis. In contrast, decrease was noted in HEK-293 cells. In MCF-7 derived cancer stem cells, fentanyl treatment decreased the expression of Bax, Bcl2, Oct4, Sox2, Nanog genes when compared to untreated cells. In HEK-293 stem cells, decrease was noted for Sox2, Nanog and Bax, but increase for Oct4. Our study supports an antitumor role of fentanyl by inducing apoptosis and reducing numbers of cancer stem cells in the MCF-7 human breast adenocarcinoma line.