Runt domain transcription factor 3 (RUNX3) is a transcription factor that functions as a tumor suppressor. RUNX3 is frequently inactivated by epigenetic silencing or its protein mislocalization (cytoplasmic localization) in many cancer types. This study investigated whether oxidative stress induces redistribution of RUNX3 from the nucleus to the cytoplasm. The cytoplasmic localization of RUNX3 was associated with oxidative stress‐induced RUNX3 phosphorylation at tyrosine residues via SRC activation. Moreover, oxidative stress increased expression of histone deacetylases (HDACs). RUNX3 phosphorylation and SRC expression induced by oxidative stress were inhibited by knockdown of HDAC1, restoring the nuclear localization of RUNX3 under oxidative stress. In conclusion, these results demonstrate that HDAC1‐ and SRC‐mediated phosphorylation of RUNX3 induced by oxidative stress is associated with the cytoplasmic localization of RUNX3 and can lead to RUNX3 inactivation and carcinogenesis. J. Cell. Physiol. 232: 1914–1921, 2017. © 2016 Wiley Periodicals, Inc. RUNX3 cytoplasmic localization induced by oxidative stress in colon cancer cells. Oxidative stress induces HDAC1 expression and then activates SRC. Activated SRC induces expression of phospho‐RUNX3. Phospho‐RUNX3 is retained in the cytoplasm, leading to degradation of cytoplasmic RUNX3 by the proteasome system and finally resulting carcinogenesis.