The inhibitory effects of limonin have been disclosed in various tumors, however, its roles in nasopharyngeal carcinoma (NPC) progression are never been revealed. In the current work, we collected NPC cells with a higher stemness compared with bulk cells through isolating the side population (SP) cells. It was found that limonin exhibited a stronger inhibitory effect on SP cells than that in bulk cells, which was evident by a lower IC50 value. Additionally, limonin attenuated the stemness and migration ability of SP cells with the higher stemness, characterized as decreasing the spheroid formation ability, expression of stemness markers and migration ability. Moreover, the proportion of SP cells in G0 phase was remarkably higher than that in bulk cells. Notably, upon limonin treatment, the proportion of SP cells in G0 was decreased and S/G2/M increased. Furthermore, limonin enhanced the radiosensitivity of NPC cells. The mechanistic studies based on RNA-sequencing analysis revealed that limonin inhibited the gene transcription driven by Stat3 (signal transducer and activator of transcription 3) and an activator of Stat3 (Colivelin or IL-6) rescued the inhibitory effects of limonin. Therefore, these results demonstrate that limonin could reduce the stemness of NPC cells and thus the radiosensitivity through suppressing Stat3 transcriptional activity.