The aim of this study was to evaluate variations of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and protein expression after adjuvant treatment in glioblastoma patients. Sixteen patients with a glioblastoma underwent 34 microsurgeries including 18 re-operations. After surgery, patients underwent follow-up with radiotherapy and chemotherapy (temozolomide, ACNU and cisplatin) between 2000 and 2008. To investigate MGMT methylation and MGMT expression, methylation-specific PCR (MSP) and immunohistochemical staining (IHC) were performed. The methylation status of the MGMT promoter was altered in five (27.8%) of 18 re-operation specimens. In four specimens, the MGMT promoter was found to be methylated after primary surgery, but was found to be unmethylated on post-treatment samples. MGMT protein expression was altered in 15 (83.3%) of 18 cases. Fifteen specimens showed higher levels of protein expression as compared to previous samples and three samples demonstrated a similar expression pattern. After irradiation and exposure to steroid and temozolomide 6 and 24 h later, a methylated MGMT promoter and negative protein expression were seen in U343 glioblastoma cell lines which have methylated promoter and negative protein expression. Variations in MGMT promoter methylation and protein expression can occur after treatment. We suggest that changes of MGMT promoter methylation and protein expression might not be related to a direct effect of irradiation and exposure to steroid and temozolomide.