Polyalthia longifolia cv. pendula (Family Annonaceae) contains active phytochemical moieties in various parts of the plant providing a valuable source for curative strategies in ailments and diseases. In the present study, the efficacy of Polyalthia longifolia leaves Methanolic Extract (PLME) was validated against two different human breast cancer cell lines-invasive ductal carcinoma cells-T-47D and adenocarcinoma cells-MDA-MB-453 cells. This reinforces the first report emphasizing the induction of apoptosis in PLME treated MDA-MB-453 cells. After 24 hours, the IC50 value of PLME was observed as 23.58 ± 3 μg/mL and 17.1 ± 1.2 μg/mL in T-47D and MDA-MB-453 cells, respectively. The early apoptotic population in PLME (20 μg/ml) treated T-47D cells was found to be 24.64% by the Annexin-V method. In MDA-MB-453 cells, 50.4% and 66.1% early apoptosis was recorded in 20 μg/ml and 40 μg/ml of PLME treated cells, respectively. The dose-dependent cell cycle arrest was observed in PLME treated T-47D and MDA-MB-453 cells. The number of cells in the G2/M phase increased, while that in the G0/G1 phase decreased in PLME treated cells. The increased concentration of PLME (40 μg/ml) significantly increases the arrest of T-47D cells in the G2/M phase (28.9%). In T-47D cells, PLME treatment resulted in a highly significant (p<0.0001 compared to untreated cells) down-regulation of BCL2. The increase in the concentration of PLME resulted in significantly increased expression of BAX and BAK1. In the current findings, the upregulation of BAK1 and BAX, cell cycle arrest, and Phosphatidylserine on the membrane of T-47D and MDA-MB-453 cells confirm apoptosis in presence of PLME.