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Objective: To investigate the effect and its underlying molecular mechanisms of essential oil from Saussurea costus in esophageal cancer cell line Eca109. Methods: The chemical composition of essential oil from Saussurea costus was investigated by gas chromatography-mass spectrometry (GC-MS). The anti-proliferative, anti-migrative, and apoptotic effects of essential oil from Saussurea costus against Eca109 cells were analyzed. Moreover, the expression of proteins associated with cell cycle, metastasis, and apoptosis was determined. Results: GC-MS analysis showed that essential oil from Saussurea costus was predominantly comprised of sesquiterpenes. Saussurea costus essential oil inhibited the viability of Eca109 cells in a dose-and time-dependent manner with IC50 values of (24.29±1.49), (19.16±2.27) and (6.97±0.86) μg/mL at 12, 24, and 48 h, respectively. The expression levels of target proteins in the cell cycle (phase G1/S), including cyclin D1, p21, and p53, were affected by Saussurea costus essential oil. The essential oil also downregulated the expression of metastasis-related proteins MMP-9 and MMP-2. Moreover, it induced apoptosis of Eca109 cells through the mitochondrial pathway, as well as inhibition of STAT3 phosphorylation. Conclusions: The essential oil from Saussurea costus exhibited anti-proliferative, anti-migrative, and apoptotic effects on Eca109 cells, and could be further explored as a potential anti-esophageal cancer agent.