Here we uncovered the involved subcellular mechanisms that sulforaphane-cysteine (SFN-Cys) inhibited invasion in human glioblastoma (GBM). SFN-Cys significantly upregulated 45 and downregulated 14 microtubule-, mitophagy-, and invasion-associated proteins in GBM cells via HPLC-MS/MS and GEO ontology analysis; SFN-Cys disrupted microtubule by ERK1/2 phosphorylation-mediated downregulation of α-tubulin and Stathmin-1 leading to the inhibition of cell migration and invasion; SFN-Cys downregulated invasion-associated Claudin-5 and S100A4, and decreased the interaction of α-tubulin to Claudin-5. Knockdown of Claudin-5 and S100A4 significantly reduced the migration and invasion. Besides, SFN-Cys lowered the expressions of α-tubulin-mediated mitophagy-associated proteins Bnip3 and Nix. Transmission electron microscopy showed more membrane-deficient mitochondria and accumulated mitophagosomes in GBM cells, and mitochondria fusion might be downregulated because that SFN-Cys downregulated mitochondrial fusion protein OPA1. SFN-Cys increased the colocalization and interplay of LC3 to lysosomal membrane-associated protein LAMP1, aggravating the fusion of mitophagosome to lysosome. Nevertheless, SFN-Cys inhibited the lysosomal proteolytic capacity causing LC3II/LC3I elevation but autophagy substrate SQSTM1/p62 was not changed, mitophagosome accumulation, and the inhibition of migration and invasion in GBM cells. These results will help us develop high-efficiency and low-toxicity anticancer drugs to inhibit migration and invasion in GBM.