Early studies suggest that TR4 nuclear receptor is a key transcriptional factor regulating various biological activities, including reproduction, cerebella development, and metabolism. Here we report that mice lacking TR4 (TR4(-/-)) exhibited increasing genome instability and defective oxidative stress defense, which are associated with premature aging phenotypes. At the cellular level, we observed rapid cellular growth arrest and less resistance to oxidative stress and DNA damage in TR4(-/-) mouse embryonic fibroblasts (MEFs) in vitro. Restoring TR4 or supplying the antioxidant N-acetyl-l-cysteine (NAC) to TR4(-/-) MEFs reduced the DNA damage and slowed down cellular growth arrest. Focused qPCR array revealed alteration of gene profiles in the DNA damage response (DDR) and anti-reactive oxygen species (ROS) pathways in TR4(-/-) MEFs, which further supports the hypothesis that the premature aging in TR4(-/-) mice might stem from oxidative DNA damage caused by increased oxidative stress or compromised genome integrity. Together, our finding identifies a novel role of TR4 in mediating the interplay between oxidative stress defense and aging.