The average survival of patients with glioblastoma is 12–15 months. Therefore, finding a new treatment method is important, especially in cases that show resistance to treatment. Extremely low-frequency electromagnetic fields (ELF-EMF) have characteristics and capabilities that can be proposed as a new cancer treatment method with low side effects. This research examines the antitumor effect of ELF-EMF on U87 and U251 glioblastoma cell lines. Flowcytometry determined the viability/apoptosis and distribution of cells in different phases of the cell cycle. The size of cells was assessed by TEM. Important cell cycle regulation genes mRNA expression levels were investigated by real-time PCR. ELF-EMF induced apoptosis in U87cells much more than U251 (15% against 2.43%) and increased G2/M cell population in U87 (2.56%,p value< 0.05), and S phase in U251 (2.4%) (data are normalized to their sham exposure). The size of U87 cells increased significantly after ELF-EMF exposure (overexpressingP53in U251 cells increased the apoptosis induction by ELF-EMF). The expression level ofP53,P21, andMDM2increased andCCNB1decreased in U87. Among the studied genes,MCM6expression decreased in U251. Increasing expression ofP53,P21and decreasingCCNB1, induction of cell G2/M cycle arrest, and consequently increase in the cell size can be suggested as one of the main mechanisms of apoptosis induction by ELF-EMF; furthermore, our results demonstrate the possible footprint ofP53in the apoptosis induction by ELF-EMF, as U87 carry the wild type ofP53and U251 has the mutated form of this gene.