The effects of methanol extract of Rubus crategifolius roots and its solvent fractions were investigated on the proliferation of MCF-7 human breast carcinoma cells. The methanol extract inhibited the proliferation of MCF-7 cells in a concentration dependent manner. Moreover, their methanol soluble (W-M) fraction had the greatest inhibitory effect on the growth of MCF-7 cells. To evaluate whether the W-M fraction affects on the cell cycle of MCF-7 cells, cells treated with this fraction were analyzed with flow cytometry. The W-M fraction increased G0/G1 phase after 24 h-treatment and induced apoptosis after 48 h-treatment. The hallmark of apoptosis, DNA fragmentation, also appeared by W-M fraction after 48 h-treatment. Furthermore, the methanol extract and its W-M fraction inhibited the activity of the topoisomerase I enzyme in the relaxation assay. From these results, their W-M fraction as well as methanol extract of R. crategifolius roots are necessary for further studies as a potent inhibitor of the growth of cancer cells.