--- - |2- Abstract The role of histone deacetylase 3 (HDAC3) is to repress the expression of various genes by eliminating acetyl group from histone. Thus, the regulation of HDAC3 activity is essential to maintain cellular homeostasis. In this study, we found that HDAC3 interacts with c‐Src kinase. However, the interaction between HDAC3 and c‐Src was previously reported, it has still been ambiguous whether c‐Src phosphorylates HDAC3 and affects the function of HDAC3. First, we confirmed that HDAC3 directly binds to c‐Src, and c‐Src identified to interact with C‐terminal domain (277–428 a.a.) of HDAC3. c‐Src also phosphorylated three tyrosine sites of HDAC3 at tyrosine 325, 328, and 331. Importantly, wild‐type c‐Src increases HDAC3 activity, but not mutant c‐SrcK298M (kinase inactive form). When these tyrosine residues are all substituted for alanine residues, the deacetylase activity of mutant HDAC3 was abolished. In addition, a proliferation of HER2‐positive breast cancer cells expressing phosphorylation deficient mutant HDAC3 is decreased in comparison with control cells. Thus, our findings suggested that phosphorylation of HDAC3 by c‐Src kinase regulates the HDAC3 activity and the proliferation of breast cancer cells. - Journal of Cellular Physiology, EarlyView.