Breast cancer is one of the most common and deadly cancers in women worldwide. Current treatments for breast cancer have limitations, such as toxicity, resistance, and side effects. Therefore, there is a need to develop new and effective anti-cancer agents from natural sources. Spinosyn A (SPA) is a natural product derived from soil bacteria. SPA has been reported to have anti-parasitic, insecticidal, and anti-bacterial activities. However, its anti-cancer effects and mechanisms are not well understood. In this study, we investigated the effects of SPA on T47-D, estrogen receptor-positive breast cancer cells. We found that SPA inhibited cell proliferation and migration and induced apoptosis and cell cycle arrest. Flow cytometry and holographic imaging microscopy revealed that SPA activated MAPK and PI3K signaling pathways and altered cellular morphology. Finally, RNA-Seq analysis revealed that SPA treatment altered the expression of 1380 genes in T47-D cells, which were enriched in various biological processes and signaling pathways related to cell proliferation, cholesterol metabolism, growth factor activity, amino acid transport activity, extracellular matrix, PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction, and PPAR signaling pathway. Our results suggest that SPA exerts multiple anti-cancer effects on T47-D cells by modulating multiple pathways and cellular processes involved in cell growth, survival, and motility. Our findings provide new insights into the molecular mechanisms of SPA action on breast cancer cells and its potential applications as a novel anti-cancer agent.