This study was earned out to investigate the antioxidative and anti-apoptotic effects of glycoprotein isolated from Gardenia jasminoides Ellis fruit (GJE glycoprotein), which has been used to heal hepatic and inflammatory diseases in folk medicine. GJE glycoprotein showed a single band with a molecular weight of 27kDa on the 15% sodium dodecyl sulfate polyacrylamide gel. It consists of a carbohydrate component (57.65%) and a protein component (42.35%). GJE glycoprotein has dose-dependent scavenging activities for DPPH, lipid peroxyl, superoxide anion and hydroxyl radicals in cell-free systems. We also evaluated the protective and anti-apoptotic activities of GJE glycoprotein on the glucose/glucose oxidase (G/GO)-induced or hypoxanthine/xanthine oxidase (HX/XO)-induced cytotoxicity and apoptosis systems in NIH/3T3 cells, using 3-(4,5-diinettiylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT), DNA fragmentation and H33342/ethidium bromide staining assays, respectively. Results in this experiment showed that GJE glycoprotein has dose-dependent blocking activities against G/GO- or HX/XO-induced cytotoxicity and apoptosis. In addition, we investigated whether GJE glycoprotein blocks the activation of redox-sensitive signal mediators, protein kinase C alpha (PKCα) and nuclear factor-kappa B (NF-κB) in G/GO or HX/XO-induced apoptotic NIH/3T3 cells, using a Western blot analysis and an electrophoretic mobility shift assay (EMSA). We found that 100μg/ml GJE glycoprotein has an inhibitory effect on PKCα translocation and the DNA binding activity of (NF-κB). Here, we speculate that GJE glycoprotein is a natural antioxidant and one of the modulators of apoptotic signal pathways in NIH/3T3 cells.