p27kip1 (p27) is an inhibitor of cyclin/cyclin-dependent kinase complexes, whose nuclear loss indicates a poor prognosis in various solid tumors. When located in the cytoplasm, p27 binds Op18/stathmin (stathmin), a microtubule (MT)-destabilizing protein, and restrains its activity. This leads to MT stabilization, which negatively affects cell migration. Here, we demonstrate that this p27 function also influences morphology and motility of cells immersed in three-dimensional (3D)matrices. Cells lacking p27 display a decrease in MT stability, a rounded shape when immersed in 3D environments, and a mesenchymal-amoeboid conversion in their motility mode. Upon cell contact to extracellular matrix, the decreased MT stability observed in p27 null cells results in accelerated lipid raft trafficking and increased RhoA activity. Importantly, cell morphology, motility, MT network composition, and distribution of p27 null cells were rescued by the concomitant genetic ablation of Stathmin, implicating that the balanced expression of p27 and stathmin represents a crucial determinant for cytoskeletal organization and cellular behavior in 3D contexts. Copyright © 2010, American Society for Microbiology. All Rights Reserved.