Pancreatic cancer, the fourth leading cause of cancer-associated mortality in the United States, usually presents in an advanced stage and is generally refractory to chemotherapy. As such, there is a great need for novel therapies for this disease. The naturally derived isoprenoids perillyl alcohol, farnesol, and geraniol have chemotherapeutic potential in pancreatic and other tumor types. However, their mechanisms of action in these systems are not completely defined. In this study, we investigated isoprenoid effects on the cell cycle and observed a similar antiproliferative mechanism of action among the three compounds. First, when given in combination, the isoprenoids exhibited an additive antiproliferative effect against MIA PaCa-2 human pancreatic cancer cells. Furthermore, all three compounds induced a G(0)/G(1) cell cycle arrest that coincided with an increase in the expression of the cyclin kinase inhibitor proteins p21(Cip1) and p27(Kip1) and a reduction in cyclin A, cyclin B1, and cyclin-dependent kinase (Cdk) 2 protein levels. Immunoprecipitation studies demonstrated increased association of both p21(Cip1) and p27(Kip1) with Cdk2 as well as diminished Cdk2 kinase activity after isoprenoid exposure, indicating a cell cycle-inhibitory role for p21(Cip1) and p27(Kip1) in pancreatic adenocarcinoma cells. When siRNA was used to inhibit expression of p21(Cip1) and p27(Kip1) proteins in MIA PaCa-2 cells, conditional resistance to all three isoprenoid compounds was evident. Given similar findings in this cell line and in BxPC-3 human pancreatic adenocarcinoma cells, we conclude that the chemotherapeutic isoprenoid compounds perillyl alcohol, farnesol, and geraniol invoke a p21(Cip1)- and p27(Kip1)-dependent antiproliferative mechanism in human pancreatic adenocarcinoma cells.