Abnormalities in cell cycle progression provide unlimited replicative potential to cancer cells, and therefore targeting of key cell cycle regulators could be a sound cancer chemopreventive strategy. Earlier, we found that grape seed extract (GSE) increases Cip/p21 protein level and inhibits growth and induces apoptosis in human colon carcinoma HT29 cells both in vitro and in vivo. However, the mechanism of GSE-induced p21 upregulation and its role in biological efficacy of GSE are not known, which were investigated here. GSE treatment of HT29 cells resulted in a strong dose- and time-dependent phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), consistent with p21 induction. The inhibition of sustained ERK1/2 activation by GSE using pharmacological inhibitors abrogated GSE-induced p21 upregulation. Furthermore, pretreatment of cells with N-acetylcysteine inhibited GSE-induced ERK1/2 phosphorylation as well as p21 upregulation, suggesting the involvement of GSE-induced oxidative stress as an upstream event. Consistent with this, GSE also decreased intracellular level of reduced glutathione. Next, we determined whether GSE-induced signaling regulates p21 expression at transcriptional and/or translational levels. GSE was found to increase the stability of p21 message with resultant increase in p21 protein level, but it did not alter the protein stability to a great extent. Importantly, knock-down of p21 abrogated GSE-induced G(1) arrest suggesting that p21 induction by GSE is essential for its G(1) arrest effect. Together, our results for the first time identify a central role of p21 induction and associated mechanism in GSE-induced cell cycle arrest in HT29 cells.