Cancer stem cells (CSCs) are a small proportion of tumor cells that may be responsible for tumor metastasis and recurrence. Our recent research indicated that longikaurin A (LK-A) exhibited anti-tumor activity in nasopharyngeal carcinoma (NPC) both in vitro and in vivo. Here, we further investigated whether LK-A could suppress the stemness of NPC cells. Sphere formation assay was used to assess the self-renewal ability of the cells treated with LK-A. Side population (SP) was determined by flow cytometry to measure the influence of LK-A on NPC SPs. The expression of the c-myc and fibronectin was detected by western blotting. The cytotoxicity of LK-A in combination with cisplatin to NPC cells was determined by MTT assay. Colony formation assay was used to verify whether LK-A could sensitize NPC cells to radiation and reverse the radiotherapy resistance. In the present study, we found that LK-A reduced the number and size of spheroid formation and decreased the SP cell percentage of the S18 cell line at a low concentration. Furthermore, LK-A treatment downregulated the expression of c-myc and fibronectin in NPC cell lines. Moreover, LK-A could significantly enhance the chemotherapeutic and radiotherapeutic sensitivity of NPC cell lines and reverse acquired radiotherapy resistance of Sune2-IR. Our data revealed that LK-A could suppress the stemness of NPC cells and may enhance the efficacy of radiotherapy and chemotherapy.