The incidence of nasopharyngeal carcinoma (NPC) in China is relatively higher than that throughout the rest of the world, and NPC is geographically distributed. Long non-coding RNA (lncRNA) plays a key role in the development of tumors. Recent studies have found that the lncRNA Ewing sarcoma-associated transcript 1 (EWSAT1) is highly expressed in various tumors and also in NPCs. The isoflavone calycosin, which is a typical Chinese herbal medicine, can inhibit the growth of breast cancer, colorectal cancer, osteosarcoma and other cancers. The aim of our study was to select NPCs that were sensitive to calycosin and whether calycosin had an effect on NPC cells. If it does, are the effects related to a high expression of EWSAT1? We also verified that EWSAT1 was highly expressed in NPC cells. At the same time, we found that calycosin inhibited the growth of NPC cell lines. To further determine whether the effect of calycosin on NPC cells was related to EWSAT1, we used NPC cells with different concentrations of calycosin and found that the expression of EWSAT1 decreased significantly with increasing concentrations of calycosin and that the expression of downstream factors and pathways were also affected. It was demonstrated that calycosin affected NPC cell growth by regulating EWSAT1 and its downstream pathway. In addition, we overexpressed EWSAT1 and found that the increased expression of EWSAT1 weakened the growth inhibitory effect of calycosin on NPC cells.