Lethariella zahlbruckneri has been traditionally used in tea and medicines in China. This study aimed to evaluate the anti-cancer properties of L. zahlbruckneri acetone extract (AEL) and to explore its potential mechanisms on MCF- 7 human breast cancer cells. The polyphenol and flavonoid concentrations of were 14.4 mg gallic acid equivalent/g and 6.5 mg quercetin equivalent/g, respectively. AEL inhibited the growth of MCF-7 cells in a dose- and time dependent manner. AEL significantly induced apoptotic cell death, resulting in an increase in the sub-G1 apoptotic cell population, apoptotic DNA fragmentation, and a morphological change. Pretreatment with a caspase inhibitor modestly attenuated the AEL-induced increase in the sub-G1 cell population, implying that caspases play a partial role in AEL-induced apoptosis. Moreover, AEL-induced apoptosis was associated with changes of caspase activities, up-regulation of the apoptotic protein (Bax), and down-regulation of the anti-apoptotic protein (Bcl-2). AEL also induced apoptosis-inducing factor-release from mitochondria, indicating apoptosis stimulation through a caspaseindependent pathway. These results suggest that AEL exerts its anti-cancer effects on MCF-7 human breast cancer cells through mitochondrial caspase-dependent and caspase-independent apoptotic pathways.