New innovations are needed for the treatment of pancreatic cancer, as current treatments do not offer significant improvements in overall survival. p21WAF1—a tumor suppressor gene—acts as a downstream effector of p53 function and mediates G1 cell cycle arrest by inhibiting cyclin-dependent kinases, which promote cell growth. p21 expression has also been shown to increase more than 20-fold in senescent cells in culture. The replication-defective recombinant adenoviral system (rAd), a major innovation in gene transfer technology, has recently been used in gene therapy applications for various malignancies but not for pancreas cancer. In this study we used rAd-p21 in cell growth inhibition studies of pancreatic tumor cell lines in vitro to explore its potential as a prospective gene therapy for pancreatic adenocarcinoma. We studied two pancreatic cell lines in culture, HPAC and Hs766T. HPAC revealed higher endogenous levels of p21 gene expression at the protein and RNA levels compared to Hs766T. p21 induction was tested using different doses of rAd-p21 to establish an optimum dose for significant induction of p21 gene expression. Tumor cell growth in culture following rAd-p21 infection was also analyzed in both cell lines. HPAC and Hs766T cell lines showed a significant dose-dependent increase in p21 protein expression when infected with rAd-p21. Both cell lines showed significant growth arrest, but Hs766T showed less cell growth inhibition than HPAC cells. Flow cytometric cell cycle analysis of rAd-p21-infected cells showed a statistically significant increase in the number of cells in G0/G1 in HPAC cells. Similar results were also obtained in Hs766T cells, however, the data were not statistically significant. In conclusion, pancreatic tumor cell growth can be inhibited by rAd-p21 in vitro, with significant numbers of tumor cells reverting from S to G0/G1. Thus rAd-p21 may be effective as a candidate gene therapy for pancreatic cancer and should be further evaluated with in vivo studies.