Cytoskeletal organization, actin-myosin contractility and the cell membrane together regulate cell morphology in response to the cell environment, wherein the extracellular matrix (ECM) is an indispensable component. Plasticity in cell shape enables cells to adapt their migration mode to their surroundings. GH3 endocrine cells respond to different ECM proteins, acquiring different morphologies: a rounded on collagen I-III (C I-III) and an elongated on collagen IV (C IV). However, the identities of the molecules that participate in these responses remain unknown. Considering that actin-myosin contractility is crucial to maintaining cell shape, we analyzed the participation of MLCK and ROCK in the acquisition of cell shape, the generation of cellular tension and the cell motility mode. We found that a rounded shape with high cortical tension depends on MLCK and ROCK, whereas in cells with an elongated shape, MLCK is the primary protein responsible for cell spreading. Further, in cells with a slow and directionally persistent motility, MLCK predominates, while rapid and erratic movement is ROCK-dependent. This behavior also correlates with GTPase activation. Cells on C I-III exhibited higher Rho-GTPase activity than cells on C IV and vice versa with Rac-GTPase activity, showing a plastic response of GH3 cells to their environment, leading to the generation of different cytoskeleton and membrane organizations and resulting in two movement strategies, rounded and fibroblastoid-like.