Breast cancer is one of the most common primary malignant tumors of among women, the long-term survival of which has stagnated in the past decades. Juglanin as a natural production mainly extracted from green walnut husks of Juglans mandshurica has been defined as the functional composition among a series of compounds. It showed powerful protective effect in various diseases by inhibiting inflammation and tumor cells growth. However, the effect of juglanin on human breast cancer and the underlying mechanisms remains to be elucidated. We reported here that juglanin could inhibit cell proliferation by leading to G2/M phase arrest. Exposure to juglanin resulted in the activation of cleaved caspase −3, −8, and −9, indicating that juglanin induced apoptosis. Autophagy occurred in juglanin-treated cells as evidenced by formation of autophagosome and up-regulation of LC3B-II. The juglanin-induced cell death was significantly restored by the combination of autophagy and apoptosis. Further, juglanin also induced JNK activation and ROS production. The JNK inhibitor attenuated juglanin-caused apoptosis and autophagy significantly while ROS scavenger could reverse them. In addition, the ROS scavenger also inhibited G2/M phase arrest and phosphorylated JNK. Of note, we found that juglanin had the similar effects on breast cancer cells. Finally, juglanin inhibited tumor growth in the mouse xenograft model in vivo. Together, our results suggested that juglanin led to G2/M phase arrest, induced apoptosis as well as autophagy through the ROS/JNK signaling pathway in human breast cancer cells. Hence, juglanin might be a promising candidate for development of anti-tumor drugs targeting breast cancer.