FM19G11 is a small molecular agent that inhibits hypoxia-inducible factor-1-alpha (HIF-1α) and other signaling pathways. In this study, we characterized the modulating effects of FM19G11 on O6 -methylguanine DNA-methyltransferase (MGMT), the main regulator of temozolomide (TMZ) resistance in glioblastomas. This study included 2 MGMT-positive cell lines (GBM-XD and T98G). MGMT promoter methylation status, mRNA abundance, and protein levels were determined before and after FM19G11 treatment, and the roles of various signaling pathways were characterized. Under hypoxic conditions, MGMT mRNA and protein levels were significantly downregulated by FM19G11 via the HIF-1α pathway in both GBM-XD and T98G cells. In normoxic culture, T98G cells were strongly positive for MGMT, and MGMT expression was substantially downregulated by FM19G11 via the NF-κB pathway. In addition, TMZ resistance was reversed by treatment with FM19G11. Meanwhile, FM19G11 has no cytotoxicity at its effective dose. FM19G11 could potentially be used to counteract TMZ resistance in MGMT-positive glioblastomas.