Objective To investigate the effect of CDKN2A/p16INK4a, a cyclin-dependent kinase inhibitor, on cell morphology and cytoskeleton in MCF7 breast cancer cells by tetracycline operon (Tet-on) inducible gene expression system. Methods MCF7 cells, genetically null for p16INK4a expression, were sequentially infected with two viruses of Tet-on systems to make MCF7-pTet-on-p16INK4a cells, which were induced to express p16NK4a by doxycycline (Dox) treatment. Cell growth was evaluated by cell counting. To observe the effect of p16INK4a on cell adhesion and cytoskeleton, intercellular adhesion and intracellular F-actin were examined by fluorescent staining with anti-E-cadherin antibody and phalloidin, respectively. Changes in cell morphology were analyzed by ImageJ software after microscopic imaging. Results Cell line expressing p16INK4a upon Dox induction was successfully made in MCF7 breast cancer cells. The inducible expression of p16INK4a inhibited cell proliferation while significantly increasing cell size as marked by prolonged contour lines. Furthermore, intracellular F-actin were enriched at the cortical peripheries of p16INK4a -expressing cells, which was different from those in control MCF7 cells where F-actin were enriched in cytoplasmic regions. Conclusion p16INK4a expression in MCF7 breast cancer cells leads to increased cell size, weakened intercellular adhesion and cortical re-distribution of cytoskeletal F-actin.