The mechanical environment in combination with biochemical signaling is an important regulatory factor for cellular physiology including tissue development, cell motility and differentiation. Exerting a tractional force triggered by myosin-dependent cell contractility is known to be an indispensible element of cell migration in a mechanically stiff environment such as a 2D planar surface. However, a number of reports have argued that the requirement of myosin activity for cell migration is limited by cell type and the environment. In this study, we present evidence that dynein, a minus end-directed microtubule motor, plays a central role in cell migration in the absence of tractional force. Interfering with the dynein activity through a dynein-specific inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), dramatically inhibited 2D migration of the fibroblast when cell contractility was blocked by Rho kinase or a myosin inhibitor, although EHNA itself did not affect cell migration. Cell migration in 3D soft collagen matrices, where the cell exerts a relatively low tractional force compared to that on a 2D stiff surface, is also profoundly inhibited by dynein intermediate chain (DIC) silencing regardless of the presence of myosin activity. In addition, DIC-silenced cells on a soft acrylamide surface show decreased migration without blockade of myosin activity. Taken together, our results suggest that dynein may be a primary regulatory factor for cell migration when a cell is in a mechanically low-tension environment, such as in a 3D matrix.