Defective fibrillar collagen polymerization in primary tumors has been correlated with increased metastasis. However, it is unclear how collagen organization influences tumor invasion. In this study, we show that collagen I polymerized without telopeptides (the flanking regions of collagen molecules) can differentially affect the three-dimensional migration of mammary carcinoma cells. MDA-MB-231 cells capable of proteolytic degradation and mesenchymal motion, invaded telopeptide-intact and telopeptide-free collagen gels to the same extent. In contrast, MDA-MB-435S cells, with typical features of amoeboid cells (poor collagenolytic activity, rounded cell morphology), were 5-fold more invasive in telopeptide-free than telopeptide-intact collagen. A fraction of the MDA-MB-435S cells that invaded telopeptide-intact or telopeptide-free collagen had a rounded morphology; however, in telopeptide-free collagen, a significant fraction of the cells switched from a rounded to elongated morphology (protrusion formation). The dynamic changes in cellular shape facilitated MDA-MB-435S locomotion through the narrow interfiber gaps, which were smaller than cell diameters. Based on the spherical morphology of MDA-MB-435S cells, we tested if the changes in cell shape and invasion were related to RhoA-ROCK activity; GTP-bound RhoA was measured in pull-down assays. RhoA activity was 1.8-fold higher for MDA-MB-435S cells seeded on telopeptide-free than telopeptide-intact collagen. Y27632 inhibition of ROCK, a Rho effector, significantly reduced the changes in cellular morphodynamics and the invasion of MDA-MB-435S cells but did not alter the invasion of MDA-MB-231 cells. Thus, the higher RhoA activity of MDA-MB-435S cells in telopeptide-free collagen enhances the changes in cellular morphodynamics associated with motility and invasion.