AIM: To investigate the antiproliferative and apoptotic effects of S-allyl cysteine (SAC) on C6 glioblastoma cells using two- and three-dimensional (2D and 3D) cell culture systems. MATERIAL AND METHODS: Three groups of rat glioma cell line C6 were prepared: 2D-Control, 2D-SAC, 3D-CMC-Control, and 3D-CMC-SAC. The control cells were incubated under standard culture conditions, the SAC cells were incubated in a culture medium supplemented with the IC50 dose (50 μM for both the 2D-SAC C6 and 3D-CMC-SAC groups) of SAC for 24 and 48 h. All experimental cells were stained with antibodies recognizing NOTCH1 and JAGGED1, and the mRNA expression levels of NOTCH1 and JAGGED1 were evaluated by qRT-PCR. RESULTS: Increasing doses of SAC were administered for 24 h to the C6 glioma cell line. The concentration of 50 μM was selected as the most suitable dose for administration. The gene expression profiles differed between these two cell culture types. We found that the expression levels of NOTCH1 receptor mRNA were lower in cells exposed to 50-μM SAC for 24 h than those of control cells in both 2D and 3D cell cultures. The immunoreactivities of both the biomarkers JAGGED1 and NOTCH1 in the glioma cells decreased significantly in the SAC group. CONCLUSION: These findings indicate that SAC is a potential drug candidate for human use, as indicated by its nontoxic nature. In addition, SAC was found to exert an anticancer effect, which is associated with the modulation of JAGGED1 and NOTCH1 signaling pathways in glioma cancer cells.