Objective: This study aimed to investigate the cytotoxicity, apoptosis induction, and mechanism of action of steviol on human breast cancer cells (Michigan Cancer Foundation-7 [MCF-7]). Materials and Methods: Sulforhodamine-B assay was performed to analyze cytotoxic potential of Steviol whereas flow cytometer was used to analyze cell cycle, apoptosis, and reactive oxygen species generation. Results: Studying the viability of cells confirms the IC50of Steviol in MCF-7 cells which was 185 μM. The data obtained from fluorescence-activated cell sorter analysis reveal Steviol-mediated G2/M-phase arrest (P < 0.05) in addition to the presence of evident sub-G0/G1 peak (P < 0.05) in the MCF-7 cells, signifying the ongoing apoptosis. Conclusion: Thus, results suggest that induction of apoptosis in MCF-7 cells was due to dose-dependent effect of Steviol. Our first in vitro findings indicate Steviol as a promising candidate for the treatment of breast cancer.