Numerous methods have been developed for preparing guiding channels/tracks to promote the alignment of highly oriented cell types. However, these manufacture methods cannot fabricate interconnected guiding channels within three-dimensional (3D) scaffolds. Providing a suitable architectural scaffold for cell attachment could lead cells to more rapidly display a desired phenotype and perform their unique functions. Previously, we developed a simple device composed of a pneumatic membrane that can generate a tunable vibration frequency to apply physical stimulation for fabricating a 3D aligned collagen fibril matrix with the characteristic D-period structure in one step. In the present study, we aimed to evaluate the cellular responses of thoracic aortic smooth muscle cells (A7r5) incorporated during the fabrication of 3D-aligned collagen fibrils with D-periods and compared these cells with those incorporated in a 3D, randomly distributed collagen matrix and in a two-dimensional (2D) aligned substrate after up to 10 days of culture. The results consistently demonstrated that A7r5 cells cultured within the 3D and 2D anisotropic matrices were aligned. Cells cultured in the 3D aligned scaffolds exhibited a higher proliferation rate as well as higher F-actin and smoothelin expression levels compared with cells cultured in 3D randomly distributed scaffolds. Together, these results indicate that a 3D-reconstituted, anisotropic collagen matrix fabricated by our process provides synergistic effects of tension stimulation and matrix stiffness on encapsulated cells and can direct A7r5 cells to transform from a synthetic phenotype into a contractile state.