Dried flowers of Trollius chinensis have long been used as an important traditional Chinese medicine. Previous studies have demonstrated the ability of T. chinensis flavonoids to reduce the proliferation of human breast cancer MCF-7 cells. The present study further investigated the influence of T. chinensis flavonoids on the growth and proliferation of MCF-7 cells and observed clear inhibitory effects within the concentration range of 0.0991–1.5856 mg/ml. Apoptosis was triggered by T. chinensis flavonoids treatment that was evaluated by differential interference contrast software, the Hoechst 33258 method, scanning electron microscopy, hematoxylin/eosin staining and laser confocal light microscopy. Cells treated with T. chinensis flavonoids selectively reduced bcl-2 and NF-κB expression and increased the expression of caspase-9 and caspase-3 indicating that the inhibition of cellular proliferation occurred through activation of a mitochondrial pathway. Taken together, the results confirmed the ability of T. chinensis flavonoids to inhibit cell proliferation.