High expression of programmed death-ligand 1 (PD-L1) in cancers is associated with low therapeutic efficacy. Sulforaphane-cysteine (SFN-Cys) is a metabolite of SFN with pro-apoptotic activity in vivo. This study aims to characterize the mechanisms that SFN-Cys causes apoptosis via downregulating PD-L1 signaling and relevant metabolic regulations in glioblastoma (GBM). Here, high expression of PD-L1 was determined in GBM tissues and was positively related to its poor prognosis. PD-L1 expression was downregulated after knockdown of α-tubulin. SFN-Cys promoted 26S proteasome-mediated degradation of α-tubulin and PD-L1, and reduced the binding of α-tubulin to PD-L1 causing apoptosis. Human leukemia monocytic cell line THP-1 was differentiated into M0-type macrophages by using phorbol-12-myristate-13-acetate (PMA). The cells were further differentiated M1- and M2-type tumor-associated macrophages (TAMs) via using conditioned medium from U87MG or U373MG cells. We found that SFN-Cys activated TAMs causing GBM cell death. More, knockdown of α-tubulin or PD-L1 decreased the expression of oncoprotein glycolytic key enzyme 6-phospho-fructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), causing NADPH reduction, reactive oxygen species (ROS) elevation, and apoptosis. Therefore, SFN-Cys might inhibit α-tubulin/PD-L1/PFKFB4 axis contributing to apoptosis, indicating its potential as an immune checkpoint blocker against human GBM.