Objective To compare the extracellular space diffusion at different stages of rat C6-gliomas determined by MRI tracer method and analyze the influencing effect of extracellular matrix (ECM) on the diffusion process.Methods Introducing adolinium-diethylene triaminepentaacetic acid (Gd-DTPA) into extracellular space (ECS) as a tracer.The diffusion parameters and half-life time were quantified according to mathematical model of diffusion. The main ECM components (e.g. chordroitin sulfate proteoglycans (CSPGs),collagen IV tenascin C) were detected by immunohistochemical and immunoblot analysis. Results Gd-DTPA introduced into 20-day glioma in the rats diffused more slowly [(6.67±1.78) ×10^-5mm²/s vs. (1.26±0.27) ×10^-4mm²/s; t=4.265; P<0.01)], deriving a larger tortuosity [(3.99±0.57) vs. (2.83±0.29); t=4.11; P<0.01)], localized within the tumor with a smaller clearance rate [(7.67±2.29) ×10^-5mm²/s)vs.(1.46±0.36) ×10^-4mm²/s);t=3.87; P<0.05), and a longer half-life time((0.86±0.23 h)vs.(1.64±0.12 h); (t=5.91; p<0.01)] compared with 10-day gliomas in the rats. The increased levels of extracellular matrix of glioma were associated with different diffusion and clearance parameters of 20-day gliomas in the rats in comparision with those in the 10-day rat gliomas, in which the chordroitin sulfate proteoglycans[(0.48±0.07) vs.(0.32±0.09);t=4.663;P<0.01)], tenascin C [(0.29±0.04) vs. (0.58±0.11);t=6.50;P<0.01] and collagen IV [(0.24±0.07)vs.(0.33±0.06);t=3.81;P<0.05] were tested. Conclusions The ECS parameters are changed with the C6 glioma progression due to the increased ECM content. The results of our study may help us to better understanding the glioma micro-environment and provide beneficial references for the brain interstitial drug delivery to treat gliomas.