By controlling DNA damage repair and regulating gene transcription, the critical epigenetic regulator histone deacetylase 3 (HDAC3) plays pivotal roles in liver cancer and liver regeneration; however, the role of HDAC3 in liver homeostasis has not been fully elucidated. Here, we found that HDAC3-deficient liver developed a defective morphology and metabolism with an increasing degree of DNA damage in the hepatocytes along the portal-central axis of the lobule. Most strikingly, the Alb-CreERT:Hdac3-/- mice showed that HDAC3 ablation did not impair liver homeostasis in terms of histological characteristics, function, proliferation, or gene profiles prior to the profound accumulation of DNA damage. We next identified that the hepatocytes in the portal area, which carried less DNA damage than those in the central area, repopulated the hepatic lobule by active regeneration and movement toward the center. As a result, the liver became more viable after each surgery. Furthermore, in vivo tracing of keratin-19 (CK19)-expressing hepatic progenitor cells (HPCs), which lacked HDAC3, showed that the HPCs gave rise to newly generated periportal hepatocytes. In hepatocellular carcinoma (HCC), HDAC3 deficiency impaired DNA damage response and enhanced radiotherapy sensitivity in vitro and in vivo. Taken together, we demonstrated that HDAC3 deficiency interferes with liver homeostasis, which is more dependent on the accumulation of DNA damage in hepatocytes than on transcriptional dysregulation. Our findings support the hypothesis that selective HDAC3 inhibition has the potential to augment the effect of chemoradiotherapy aimed at inducing DNA damage in cancer therapy.Copyright © 2023. Published by Elsevier Inc.