Thunbergia Laurifolia Linn. (TL) is one of the most familiar plants in Thai traditional medicine that is usedto treat various conditions, including cancer. However, the antitumor activity of TL or its constituents has neverbeen reported at the molecular level to support the folklore claim. The present study was designed to investigatethe antitumor effect of an aqueous extract of TL in human breast cancer cells and the possible mechanism(s) ofaction. An aqueous crude extract was prepared from dried leaves of TL. Folin-Ciocalteu colorimetric assays wereused to determine the total phenolic content. Antiproliferative and cell cycle effects were evaluated in humanbreast adenocarcinoma MCF-7 cells by MTT reduction assay, cell growth inhibition, clonogenic cell survival,and flow cytometric analysis. Free radical generation by the extracts was detected using electron paramagneticresonance spectroscopy. The exposure of human breast adenocarcinoma MCF-7 cells to a TL aqueous extractresulted in decreases in cell growth, clonogenic cell survival, and cell viability in a concentration-dependentmanner with an IC50 value of 843 μg/ml. Treatments with extract for 24h at 250 μg/ml or higher induced cellcycle arrest as indicated by a significant increase of cell population in the G1 phase and a significant decreasein the S phase of the cell cycle. The capability of the aqueous extract to generate radical intermediates wasobserved at both high pH and near-neutral pH conditions. The findings suggest the antitumor bioactivities ofTL against selected breast cancer cells may be due to induction of a G1 cell cycle arrest. Cytotoxicity and cellcycle perturbation that are associated with a high concentration of the extract could be in part explained by thetotal phenolic contents in the extract and the capacity to generate radical intermediates to modulate cellularproliferative signals.