To investigate the mechanism of cell apoptosis in human esophageal carcinoma cells Eca109 induced by 85% ethanol elution fraction of Artemisia absinthium L. extract with macroporous resin(AEMV), in this study, the effect on the proliferation of Eca109 cells induced by AEMV was detected by MTT assay in vitro. Flow cytometry was used to detect the change of cell cycle and apoptosis ratio. The change of related proteins expression was detected by Western Blot. The results showed that AEMV could significantly inhibit cell proliferation in a concentration dependent manner, which compared with the DMSO group(P <0.001). The IC50 is 57.76 μg/mL. AEMV could induce apoptosis(P <0.001) and chromatin condensation of Eca109 cells. The cell cycle was arrested by down regulating the expression of cyclin B1. 50 μg/mL AEMV induced arrest cell cycle at the G0/G1 phase, 100 μg/mL AEMV induced cell cycle arrest at the G2/M phase. 100 μg/mL AEMV can increase the level of intracellular reactive oxygen species(P <0.001), decrease the mitochondrial membrane potential(P <0.001) and activate the mitochondrial-mediated intrinsic caspase-dependent pathway by increasing the expression of cleaved caspase-9, caspase-3 and PARP in a dose-dependent manner. This study also found that AEMV could activate endoplasmic reticulum stress(ER stress) in Eca109 cells by up regulating the expression of GRP78, p-PERK, p-eIF-2α and CHOP. In conclusion, AEMV induces ROS production, activates endoplasmic reticulum stress and induces apoptosis of Eca109 cells through mitochondrial-mediated intrinsic caspase-dependent pathway. It is suggested that AEMV may be a potential candidate drug for the treatment of esophageal carcinoma.