Cell adhesion and orientation are important for both natural and engineered tissues to fully achieve physiologic functions. Based on diverse cellular responses induced by electrical stimulus on 2D substrate, we applied non-invasive electrical stimulus to regulate cell adhesion and orientation of bone marrow-derived mesenchymal stem cells (MSCs) and fibroblasts in a reconstituted 3D collagen-based scaffold. While fibroblasts were induced to reorient perpendicularly in response to direct current electrical stimulus, rat MSCs showed only slight changes in cell reorientation. Multiphoton microscopy revealed that rat MSCs exhibited much stronger 3D adhesion, which appears to resist cell reorientation. Only in response to a large electrical stimulus (e.g., 10 V/cm), collagen fibers around rat MSCs became disconnected and loosely reorganized. In contrast, the collagen fibers surrounding the fibroblasts were entangled in a random network and became preferentially aligned in the direction of the electrical stimulus. When incubated with integrin antibodies, both fibroblasts and rat MSCs failed to respond to electrical stimulus, providing evidence that integrin-dependent molecular mechanisms are involved in 3D cell adhesion and orientation. Elucidation of physical regulation of 3D cell adhesion and orientation may offer a novel approach in controlling cell growth and differentiation and could be useful for stem cell-based therapeutic application and engineering tissue constructs.