Background: Metalloproteinase-9 (MMP-9) and aquaporin-4 (AQP4) have been individually reported in glioma development. Here, we co-analyzed their expression in multiple forms of human glioma tissues graded from II to IV. Material and methods: Levels of MMP-9 and AQP4 were evaluated on 50 resected human glioma tissues using immunohistochemistry. Protein levels of both molecules were evaluated by a staining score system based on the percentage of positive cells/staining degree in each dot section. The transwell method was also used to discriminate fast migrating cells and slow migrating cells, in which expression of both MMP-9 and AQP4 was investigated by using immunofluorescence. Results: The staining score of MMP-9 displayed a positively tumor grade dependent manner, whereas AQP4 expression showed a negatively tumor grade dependent manner The nuclear translocation of both molecules was observed in astrocytomas with glioblastoma transition, or glioblastoma tissues. Fast migrating cells contain more AQP4, whereas more MMP-9 was localized in slow migrating cells. Conclusions: Our findings suggest differential expression patterns of MMP-9 and AQP4 in different grades of gliomas. Nuclear translocation of MMP-9 and AQP4 may exert more functions in glioblastoma transition or deterioration. Co-analysis of MMP-9 and AQP4 may help to identify tumor type and their progression stages.