Inter- and intratumoral heterogeneity is a hallmark of glioblastoma (GBM) that facilitates recurrence, treatment resistance, and worse prognosis. O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is a significant prognostic marker for Temozolomide (TMZ) resistance in GBM patients.YKL-40is a molecular marker for the mesenchymal subtype of GBMs and is responsible for TMZ resistance. However, underlying mechanisms by which MGMT epigenetics impacts patient outcomes and the function of YKL-40 are not fully determined. Herein, we performed in vitro and in vivo experiments, six humanIDH1/2wild-type glioblastoma stem-like cells (GSCs) were established and studied to further determine a potential interaction of YKL-40 and MGMT promoter methylation. We demonstrated thatYKL-40functioned differently in humanIDH1/2wild-type GSCs. InMGMTpromoter-methylated (MGMT-m) GSCs, it acted as a tumor suppressor gene. On the other hand, inMGMTpromoter-unmethylated (MGMT-um) GSCs, it promoted tumorigenesis. Notably, the reason thatYKL-40played different roles in GSCs could not be interpreted by the molecular classification of each GSCs, but is a function ofMGMTpromoter methylation status and involves theRAS-MEK-ERKpathway.YKL-40mediated TMZ sensitivity by activating DNA damage responses (DDRs) inMGMT-mGSCs, and it mediated resistance to TMZ by inhibiting DDRs inMGMT-umGSCs. Our report demonstrated thatMGMTpromoter methylation status might influence a gene's function in human cancer. Moreover, our data also highlight the point that gene function should be investigated not only according to the molecular tumor classification, but also the epigenetic signature.