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The anticancer activity of Ocimum basilicum extract and its fractions was evaluated using human cancer cell lines; active compound(s) residing in it were identified and mechanism of their anti-proliferative action was explored. Methanolic extract was fractionated into petroleum ether soluble (PE-S) and insoluble (PE-I) fractions. These were evaluated on HT-144, MCF-7, NCI-H460 and SF-268 cell lines using Sulforhodamine B assay. Immunofluorescence microscopy was employed to study their effects on the cytoskeleton and nuclei of MCF-7 cells. Fractionation of PE-I (GI50: 5 μg/ml; LC50: 71μg/ml against MCF-7) led to the isolation of four compounds, mainly ursolic acid (LC50: 18.6 μg/ml). Ursolic acid (100 μM) induced a significant decrease in the percentage of cells in anaphase/telophase stages along with F-actin aggregation and mitotic spindle distortion. These results support anti-proliferative activity of O. basilicum extract against MCF-7 cells which may partly be due to effects of ursolic acid on F-actin and microtubules.