Aims We aimed to investigate the role of receptor‐interacting protein 2 (RIP2) in regulation of stemness of glioma cells and chemotherapy resistance. Methods Plasmid transfection was used to overexpress RIP2. Chemical inhibitors were used to inhibit RIP2 or NF‐κB activity. Cancer stemness of glioma cells was investigated by sphere formation assays, clone formation assays, and xenograft tumor formation assays. The expression of RIP2, p‐NF‐κB, IκBα, CD133, or SOX‐2 was detected by Western blotting and immunofluorescence. Apoptosis was detected by flow cytometry. Immunohistochemical staining was used to detect the expression of RIP2, CD133, and SOX‐2 in xenograft tumor tissue. The effect of the RIP2/NF‐κB pathway on temozolomide (TMZ) resistance was evaluated by xenograft tumor assay. Results Transfection with RIP2 plasmid enhanced the sphere formation capability of U251 cells, clone formation capability, and xenograft tumor formation capability. RIP2 could mediate TMZ resistance by upregulating the expression of CD133 and SOX‐2 by activating the NF‐κB pathway. Both RIP2 inhibitor GSK583 and the NF‐κB inhibitor SC75741 could reverse the resistance of U251 cells to TMZ. Conclusion RIP2 mediates TMZ resistance by regulating the maintenance of stemness in glioma cells through NF‐κB. Interventions targeting the RIP2/NF‐κB pathway may be a new strategy for TMZ‐resistant gliomas. RIP2 is involved in the induction of TMZ resistance in gliomas. The RIP2/NF‐κB signaling pathway induces enhanced stemness and plays an important role in TMZ resistance. The combined application of RIP2 or NF‐κB inhibitors and TMZ may be a new strategy for the treatment of RIP2‐related drug‐resistant gliomas.