Skin aging is characterized by wrinkle formation and increased frailty and laxity, leading to the risk of age-related skin disease. The keratinocyte is an important component of the epidermis in skin structure, and keratinocyte senescence has been identified as a pivotal factor in skin aging development. Because epigenetic pathways play a vital role in the regulation of skin aging, we evaluated human skin samples for DNA hydroxymethylation (5-hydroxymethylcytosine; 5-hmC) and SIRT4 expression. Results found that both 5-hmC and SIRT4 showed a significant decrease in aged human skin samples. To test the results in vitro, human keratinocytes were cultured in H2O2, which modulates skin aging in vivo. H2O2-induced keratinocytes showed senescence-associated protein expression as well as significant downregulation of 5-hmC and SIRT4 expression. Moreover, 5-hmC converting enzymes ten-eleven translocation 2 (TET2) showed a decrease as well as enhanced TET2 acetylation level in H2O2-induced keratinocytes. However, the overexpression of SIRT4 in keratinocyte alleviate the senescence phenotype such as senescence-associated protein expression as well as decrease the TET2 acetylation but increase TET2 and 5-hmC expression. Our results provide a novel relevant mechanism whereby epigenetic regulation of keratinocytes in skin aging may be correlated with SIRT4 expression and TET2 acetylation in 5-hmC alteration. Our study may provide a potential strategy for anti-skin aging, which targets the SIRT4/TET2 axis involving epigenetic modification in keratinocyte senescence.Copyright © 2023. Published by Elsevier Inc.