One of the insidious biological features of gliomas is their potential to extensively invade normal brain tissue, yet molecular mechanisms that dictate this locally invasive behavior remain poorly understood. To investigate the molecular basis of invasion by malignant gliomas, proteomic analysis was performed using a pair of canine glioma subclones – J3T‐1 and J3T‐2 – that show different invasion phenotypes in rat brains but have similar genetic backgrounds. Two‐dimensional protein electrophoresis of whole‐cell lysates of J3T‐1 (angiogenesis‐dependent invasion phenotype) and J3T‐2 (angiogenesis‐independent invasion phenotype) was performed. Twenty‐two distinct spots were recognized when significant alteration was defined as more than 1.5‐fold change in spot intensity between J3T‐1 and J3T‐2. Four proteins that demonstrated increased expression in J3T‐1, and 14 proteins that demonstrated increased expression in J3T‐2 were identified using liquid chromatography‐mass spectrometry analysis. One of the proteins identified was annexin A2, which was expressed at higher levels in J3T‐1 than in J3T‐2. The higher expression of annexin A2 in J3T‐1 was corroborated by quantitative RT‐PCR of the cultured cells and immunohistochemical staining of the rat brain tumors. Moreover, immunohistochemical analysis of human glioblastoma specimens showed that annexin A2 was expressed at high levels in the tumor cells that formed clusters around dilated vessels. These results reveal differences in the proteomic profiles between these two cell lines that might correlate with their different invasion profiles. Thus, annexin A2 may be related to angiogenesis‐dependent invasion.