Our previous study has shown that ampelopsin (AMP), a flavonol mainly found in Ampelopsis grossedentata, could induce cell death in human breast cancer cells via reactive oxygen species generation and endoplasmic reticulum (ER) stress pathway. Here, we examined whether autophagy is activated in AMP-treated breast cancer cells and, if so, sought to find the exact role and underlying molecular profile of autophagy in AMP-induced cell death. Our results showed that AMP treatment activated autophagy in MDA-MB-231 and MCF-7 breast cancer cells, as evidenced by the accumulation of autophagosomes, an increase of microtubule-associated protein 1 light chain 3 beta-2 (LC3B-II) and the conversion of LC3B-I to LC3B-II, the degradation of the selective autophagic target p62/SQSTM1, and the formation of green fluorescent protein (GFP)-LC3 puncta. Blockage of autophagy augmented AMP-induced cell death, suggesting that autophagy has cytoprotective effects. Meanwhile, AMP treatment suppressed Akt-mammalian target of rapamycin (mTOR) pathway as evidenced by dose- and time-dependent decrease of the phosphorylation of Akt, mTOR and ribosomal protein S6 kinase (p70S6K), whereas Akt activator insulin-like growth factor-1 (IGF-1) pretreatment partially restored Akt-mTOR pathway inhibited by AMP and decreased AMP-inuduced autophagy, signifying that AMP activated autophagy via inhibition of the Akt-mTOR pathway. Additionally, blocking ER stress not only reduced autophagy induction, but also alleviated inhibition of the Akt-mTOR pathway induced by AMP, suggesting that activation of ER stress was involved in induction of autophagy and inhibition of the Akt-mTOR pathway. Taken together, these findings indicate that AMP induces protective autophagy in human breast cancer cells through Akt-mTOR pathway via ER stress.