An elevated level of glucose has been found in the blood of hyperglycemia and diabetes patients associated with several central nervous system (CNS) complications. These disorders may be due to the up-regulation of many neurotoxic mediators by host cells triggered by high glucose (HG). Moreover, increased plasma levels of matrix metalloproteinases (MMPs), MMP-9 especially, have been observed in patients with brain injuries and may contribute to brain diseases. However, the HG level triggers the CNS pathological responses during the hyperglycemia and diabetes remain unclear. In this study, we use a transformed astroglial cell (RBA-1) as a model to investigate the signaling mechanisms of MMP-9 induction by HG and its effects on neuronal cells. First, the data by gelatin zymographic and Western blotting analyses demonstrated that HG-induced MMP-9 expression. Next, the data obtained with selective pharmacological inhibitors and small interfering RNAs showed that HG-induced MMP-9 expression via a c-Src-dependent transactivation of platelet-derived growth factor receptor and PI3K/Akt linking to NADPH oxidase 2-derived reactive oxygen species signal and activation of MAPKs. Subsequently, the transcriptional factor AP-1 was activated and thereby turned on transcription of MMP-9 gene. Ultimately, we found that HG-induced MMP-9 expression from astrocytes resulted in neuronal apoptosis. These results will provide new insights into the mechanisms and effects of the action of HG, supporting the hypothesis that HG may cause brain disorders in the development of diabetes and hyperglycemia-induced CNS complications such as neurodegenerative diseases.