Esophageal cancer is one of the leading cause of cancer mortality in the world. Due to the increased drug and radiation tolerance, it is urgent to develop novel anticancer agent that triggers nonapoptotic cell death to compensate for apoptosis resistance. In this study, we show that treatment with gypenoside L (Gyp-L), a saponin isolated from Gynostemma pentaphyllum, induced nonapoptotic, lysosome-associated cell death in human esophageal cancer cells. Gyp-L-induced cell death was associated with lysosomal swelling and autophagic flux inhibition. Mechanistic investigations revealed that through increasing the levels of intracellular reactive oxygen species (ROS), Gyp-L triggered protein ubiquitination and endoplasm reticulum (ER) stress response, leading to Ca2+ release from ER inositol trisphosphate receptor (IP3R)-operated stores and finally cell death. Interestingly, there existed a reciprocal positive-regulatory loop between Ca2+ release and ER stress in response to Gyp-L. In addition, protein synthesis was critical for Gyp-L-mediated ER stress and cell death. Taken together, this work suggested a novel therapeutic option by Gyp-L through the induction of an unconventional ROS-ER-Ca2+-mediated cell death in human esophageal cancer.