Fisetin, a natural flavonol present in edible vegetables, fruits, and wine, was reported to exert anticarcinogenic effects. The objective of the current study was to examine the effect of fisetin on the cell cycle progression of the human colon cancer cell line HT-29. HT-29 cells were cultured in serum-free medium with 0, 20, 40, or 60 µmol/L fisetin. Fisetin dose dependently inhibited both cell growth and DNA synthesis (P < 0.05), with a 79 ± 1% decrease in cell number observed 72 h after the addition of 60 µmol/L fisetin. Perturbed cell cycle progression from the G1 to S phase was observed at 8 h with 60 µmol/L fisetin treatment, whereas a G2/M phase arrest was observed after 24 h (P < 0.05). The phosphorylation state of the retinoblastoma proteins shifted from hyperphosphorylated to hypophosphorylated in cells treated with 40 µmol/L fisetin. (P < 0.05). Fisetin decreased the activities of cyclin-dependent kinases (CDK)2 and CDK4; these effects were likely attributable to decreases in the levels of cyclin E and D1 and an increase in p21CIP1/WAF1 levels (P < 0.05). However, fisetin also inhibited CDk4 activity in a cell-free system (P < 0.05), indicating that it may directly inhibit CDk4 activity. The protein levels of cell division cycles (CDC)2 and CDC25C and the activity of CDC2 were also decreased in fisetin-treated cells (P < 0.05). These results indicate that inhibition of cell cycle progression in HT-29 cells after treatment with fisetin can be explained, at least in part, by modification of CDK activities.