Abstract: Zuojinwan is a traditional Chinese medicine, and the important role of water extract of Zuojinwan in breast cancer progression was shown. Zuojinwan significantly inhibited cell proliferation, migration and invasion in triple-negative breast cancer (TNBC) cells, however, it had no significant effect on normal breast epithelial cells. Additionally, the effect of Zuojinwan on TNBC may involve inducing cell autophagy by upregulation of ATG4B. Moreover, autophagy induction suppressed TNBC progression. The knockout of ATG4B abolished the Zuojinwan induced autophagy and significantly reduced the inhibitory effect of Zuojinwan on TNBC, which suggests that ATG4B is essential for the Zuojinwan-induced autophagy in the TNBC cells. Therefore, Zuojinwan has a significant anti-cancer effect on TNBC by inducing ATG4B-mediated autophagy, which may provide a potential strategy for the treatment of TNBC.