Diabetes manifests from a loss in functional beta cell mass, which is regulated by a dynamic balance of various cellular processes including beta cell growth, proliferation, and death as well as secretory function. The cell cycle machinery comprised of cyclins, kinases, and inhibitors regulate proliferation. However, their involvement during beta cell stress during the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two pharmacologic inducers of beta cell stress, dexamethasone and thapsigargin. We hypothesized that beta cell stress upregulates p21 to activate the apoptotic pathway and suppress cell survival signaling. To this end, p21 was adenovirally overexpressed in pancreatic rat islets and 832/13 cells. As expected, p21 overexpression resulted in decreased [3H]-thymidine incorporation. Flow cytometry analysis in p21-transduced 832/13 cells verified lower replication as indicated by a decreased cell population in S-phase and a block in G2/M transition. The sub-G0 cell population was higher with p21 overexpression and was attributable to apoptosis, demonstrated by increased annexin-positive stained cells and cleaved caspase 3 protein. p21-mediated caspase 3 cleavage was inhibited by either overexpression of the anti-apoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the pro-apoptotic proteins Bax and Bak. Therefore, an intact intrinsic apoptotic pathway is central for p21-mediated cell death. In summary, our findings indicate that beta cell apoptosis can be triggered by p21 during stress, thus being a potential target to inhibit for protection of functional beta cell mass.