Temozolomide (TMZ) is the leading chemotherapeutic agent used for glioma therapy due to its good oral absorption and blood-brain barrier permeability. However, its anti-glioma efficacy may be limited due to its adverse effects and resistance development. O6-Methylguanine-DNA-methyltransferase (MGMT), an enzyme associated with TMZ resistance, is activated via the NF-κB pathway, which is found to be upregulated in glioma. TMZ also upregulates NF-κB signaling like many other alkylating agents. Magnolol (MGN), a natural anti-cancer agent, has been reported to inhibit NF-κB signaling in multiple myeloma, cholangiocarcinoma, and hepatocellular carcinoma. MGN has already shown promising results in anti-glioma therapy. However, the synergistic action of TMZ and MGN has not been explored. Therefore, we investigated the effect of TMZ and MGN treatment in glioma and observed their synergistic pro-apoptotic action in both in vitro and in vivo glioma models. To explore the mechanism of this synergistic action, we found that MGN inhibits MGMT enzyme both in vitro and in vivo glioma. Next, we established the link between NF-κB signaling and MGN-induced MGMT inhibition in glioma. MGN inhibits the phosphorylation of p65, a subunit of NF-κB, and its nuclear translocation to block NF-κB pathway activation in glioma. MGN-induced NF-κB inhibition results in the transcriptional inhibition of MGMT in glioma. TMZ and MGN combinatorial treatment also impedes p65 nuclear translocation to inhibit MGMT in glioma. We observed a similar effect of TMZ and MGN treatment in the rodent glioma model. Thus, we concluded that MGN potentiates TMZ-induced apoptosis in glioma by inhibiting NF-κB pathway-mediated MGMT activation. [Display omitted] • Magnolol potentiates the anti-glioma efficacy of Temozolomide. • Magnolol inactivates NF-κB pathway by blocking p65 nuclear translocation and inhibits MGMT in glioma. • Magnolol and Temozolomide exert synergistic apoptosis in glioma due to MGMT inhibition by Magnolol. [ABSTRACT FROM AUTHOR]