MicroRNA-21 (miR-21) is overexpressed virtually in all human cancers and displays oncogenic activity in a transgenic murine model. Similarly, the p53 tumor suppressor gene is the most frequently mutated gene in human cancer, and its loss or mutation leads to tumor formation in mice. To ascertain the role of miR-21 in the p53 pathway in vivo and to characterize their interaction in tumorigenesis, we intercrossed the miR-21 (-/-) and Trp53 (-/-) mice. We found that Trp53 (-/-) miR-21 (-/-) mice develop tumors at a slightly later age, yet show a similar tumor spectrum and survival curve as Trp53 (-/-) mice. When subjected to genotoxic agents, tissues from Trp53 (-/-) miR-21 (-/-) mice have a higher percentage of apoptotic cells. We extracted mouse embryonic fibroblast cells (MEFs) to examine the impact of miR-21 loss on p53-regulated cellular processes in Trp53 (-/-) cells. Higher cellular apoptosis and senescence were found in Trp53 (-/-) miR-21 (-/-) MEFs than in Trp53 (-/-) MEFs. In addition, loss of miR-21 sensitizes transformed Trp53 (-/-) cells to DNA damage-induced apoptosis through elevation of Pten expression. These data suggest that inhibition of miR-21 would be beneficial in apoptosis-inducing cancer therapies directed against p53-deficient tumors.