BACKGROUND: Sulforaphane (SFN), a naturally occurring isothiocyanate derived from cruciferous vegetables, has shown promise as a multitargeted therapeutic agent in glioblastoma (GBM). This study aimed to elucidate the role and underlying molecular mechanisms of SFN in regulating GBM progression, particularly through the endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) pathways. METHODS: Primary human glioma cells and established GBM cell lines were treated with various concentrations of SFN. RNA sequencing and qPCR analyses were conducted to identify transcriptional changes associated with the UPR pathway. Western blot and immunofluorescence were used to assess the expression and subcellular localization of key ER stress-related proteins. A CHOP knockdown model was employed to examine the functional role of CHOP in SFN-induced apoptosis. Additionally, normal human astrocytes (HA) were used to evaluate the selectivity of SFN's cytotoxicity. In vivo validation was performed using an intracranial glioma xenograft mouse model. RESULTS: SFN significantly induced apoptotic cell death in GBM cells. Mechanistically, SFN activated multiple branches of the UPR, notably increasing the expression and nuclear translocation of ATF4 and CHOP. CHOP knockdown markedly attenuated SFN-induced apoptosis. RNA-seq and KEGG enrichment analyses confirmed the involvement of the ER stress pathway. Treatment with 4-phenylbutyrate (4-PBA) suppressed SFN-induced cytotoxicity, further supporting ER stress-mediated apoptosis. In vivo, SFN reduced tumor burden and upregulated ER stress markers in intracranial tumor tissues. Importantly, SFN had minimal cytotoxic effects on normal astrocytes, suggesting a favorable therapeutic window. CONCLUSIONS: This study demonstrates that SFN induces GBM cell apoptosis via activation of the UPR pathway, particularly through the ATF4-CHOP axis. These findings support the potential of SFN as a promising therapeutic agent for glioblastoma.