Phenethyl Isothiocyanate (PEITC) is a well-studied compound within the isothiocyanate family. Accumulating evidence indicates that PEITC induces apoptosis and inhibits the growth of various cancer cells in vitro, including aggressive glioblastoma cells. However, its tumor suppression effects and mechanisms in vivo remain largely unexplored. In this study, we utilized cell culture experiments and an orthotopic transplant brain tumor model in mice to evaluate the impact of PEITC on tumor growth, physiological changes, and immune cell populations. Our results showed that PEITC significantly reduced the viability of glioma cells while having moderate effects on astrocytes. In vitro, PEITC effectively inhibited cell viability, migration, and invasion in GL-261 cells. In vivo, PEITC treatment led to prolonged survival rates and reduced tumor volumes in mice without significant toxicity. Notably, PEITC increased the populations of natural killer (NK) cells and natural killer T (NKT) cells in peripheral blood, indicating an immunomodulatory effect. Migration Inhibitory Factor (MIF) was identified as a potential direct target of PEITC. Our findings revealed that PEITC significantly reduced MIF expression in GL-261 cells, both in culture and in orthotopic tumor tissue, and decreased MIF-induced cellular signaling. These results suggest that PEITC has potential to be a therapeutic agent for glioblastoma by inhibiting tumor growth and modulating the immune response through MIF suppression.