Eupatilin, a type of flavonoid compound, has potential anti‑inflammatory and antitumor roles in gastric cancer and endometrial cancer; however, the effect of eupatilin on human esophageal cancer and the underlying molecular mechanisms remain unclear. In the present study, we investigated the antitumor effect of eupatilin on human esophageal cancer cells in vitro and in vivo. We found that eupatilin inhibited the proliferation and colony formation of esophageal cancer TE1 cells. DNA content analysis showed that eupatilin induced cell cycle arrest of TE1 cells at the G0/G1 phase. In addition, our results suggested that eupatilin suppressed TE1 cell proliferation by targeting the Akt/GSK3β and MAPK/ERK signaling cascades. Furthermore, treatment with eupatilin was found to decrease tumor volume in a TE1 xenograft mouse model, and the phosphorylation of Akt and ERK1/2 was inhibited by eupatilin in the tumor tissue. Notably, no obvious weight loss for the mice was detected. In conclusion, the present results indicate that the antiproliferative effect of eupatilin on esophageal cancer TE1 cells is associated with inhibition of the Akt and ERK pathways.