The levels of expression of O6-methylguanine-DNA methyltransferase (MGMT) are relevant in predicting the response to the alkylating chemotherapy in patients affected by glioblastoma. MGMT promoter methylation and the published MGMT regulating microRNAs (miRNAs) do not completely explain the expression pattern of MGMT in clinical glioblastoma specimens. Here we used a genome-wide microarray-based approach to identify MGMT regulating miRNAs. Our screen unveiled three novel MGMT regulating miRNAs, miR-127-3p, miR-409-3p, and miR-124-3p, in addition to the previously identified miR-181d-5p. Transfection of these three novel miRNAs into the T98G glioblastoma cell line suppressed MGMT mRNA and protein expression. However, their MGMT- suppressive effects are 30-50% relative that seen with miR-181d-5p transfection. In silico analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) revealed that miR-181d-5p is the only miRNA that consistently exhibited inverse correlation with MGMT mRNA expression. However, statistical models incorporating both miR-181d-5p and miR-409-3p expression better predict MGMT expression relative to models involving either miRNA alone. Our results confirmed miR-181d-5p as the key MGMT-regulating miRNA. Other MGMT regulating miRNAs, including the miR-409-3p identified in this report, modify the effect of miR-181d-5p on MGMT expression. MGMT expression is, thus, regulated by cooperative interaction between key MGMT-regulating miRNAs.