In this study, eleven new 3- and 7-positions modified scopoletin derivatives (18a-k) were designed, synthesized, and biologically evaluated against human breast cancer cell lines. Most compounds showed improved antiproliferative activity against MCF-7 and MDA-MB-231 cells and weaker cytotoxicity on human breast epithelial cell line MCF-10A than lead compound 5. Among them, compound 18e exhibited the most potent antiproliferative activity against MCF-7 cells (IC<sub>50</sub> = 0.37 ± 0.05 μM). Particularly, 18e produced the highest levels of nitric oxide (NO) intracellularly, and its antiproliferation effect was attenuated by hemoglobin (an NO scavenger). Further pharmacological research showed that 18e blocked the cell cycle at the G<sub>2</sub>/M phase, downregulated the phosphorylation of PI3K and Akt in MCF-7 cells and regulated the expressions of the apoptosis proteins to induce apoptosis. Moreover, 18e inhibited the growth of MCF-7 in vivo. Overall, 18e is a novel anticancer agent with the abilities of high concentration of NO releasing and the inhibition of PI3K/Akt signaling pathway, and may be a promising agent against MCF-7 human breast cancer.