The migration and proliferation of keratinocytes are critical for re-epithelization during chronic wound healing. Runt-related transcription factor 1 (RUNX1) has been indicated to repress keratinocyte proliferation. Nonetheless, the potential molecular mechanism of RUNX1 in regulating keratinocyte proliferation and migration remains unclear. CCK-8 and wound healing assays were implemented for examining keratinocyte viability and migration, respectively. Western blotting and RT-qPCR were utilized for quantifying protein and RNA levels. Luciferase reporter assay was employed for verifying the interaction between RUNX1, miR-17-5p and lncRNA H19. The results showed that RUNX1 depletion promoted keratinocyte proliferation and migration and repressed extracellular matrix degradation. Mechanistically, H19 upregulated RUNX1 expression by competitively absorbing miR-17-5p. Rescue experiments revealed that overexpressing RUNX1 reversed H19 silencing-mediated effects on the phenotypes of keratinocytes. In conclusion, H19 knockdown promotes keratinocyte proliferation and migration and suppresses extracellular matrix degradation via the miR-17-5p/RUNX1 axis.© The Author(s) 2023. Published by Oxford University Press on behalf of the American Burn Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.