Keratin filaments impart resilience against mechanical extension of the cell. Despite the pathophysiological relevance of this function, very little is known about the mechanical properties of intermediate filaments in living cells and how these properties are modulated. We used keratin mutants that mimic or abrogate phosphorylation of keratin 8‐serine⁴³¹ and keratin 18‐serine⁵² and investigated their effect on keratin tortuousness after cell stretch release in squamous cell carcinoma cells. Cells transfected with the wild‐type keratins were used as controls. We can show that keratin dephosphorylation alters the stretch response of keratin in living cells since keratin tortuousness was abolished when phosphorylation of keratin18‐serine⁵² was abrogated. Additional experiments demonstrate that keratin tortuousness is not simply caused by a plastic overextension of keratin filaments because tortuousness is reversible and requires an intact actin‐myosin system. The role of actin in this process remains unclear, but we suggest anchorage of keratin filaments to actin during stretch that leads to buckling on stretch release. Dephosphorylated keratin18‐serine⁵² might strengthen the recoil force of keratin filaments and hence explain the abolished buckling. The almost exclusive immunolabeling for phosphorylated keratin18‐serine ⁵² in the cell periphery points at a particular role of the peripheral keratin network in this regard.—Fois, G., Weimer, M., Busch, T., Felder, E. T., Oswald, F., von Wichert, G., Seufferlein, T., Dietl, P., Felder, E. Effects of keratin phosphorylation on the mechanical properties of keratin filaments in living cells. FASEB J. 27, 1322–1329 (2013). www.fasebj.org