Although structural anisotropy is important for proper function of normal myocardium, it has been a challenge to mimic structural architecture of native tissue in vitro. Previous studies have focused on using two-dimensional cultures utilizing micropatterning of substrates. Here, we present three-dimensional (3D) decellularized engineered tissues with a controlled varying degree of extracellular matrix alignment. Engineered tissues were initially created by embedding cardiac fibroblasts in 3D collagen tissues (rat tail type I collagen, BD sciences) and cultured either under biaxial or uniaxial constraints. Biaxially constrained tissues yielded a random orientation of both cell and collagen fibers, while uniaxially constrained tissues showed that the cell and collagen fibers aligned parallel to the constrained direction. To facilitate cardiac fibroblast deposition of extracellular matrix, culture medium was supplemented with 5 ng/ml ascorbic acid every 3 days. Once desired structural alignment was obtained, cells were removed by decellurization treatment using CHAPS buffer followed by SDS buffer for 1 hr at 37°C with gentle agitation. Removal of cells was confirmed by DNA quantification and intact collagen fiber orientation was confirmed by confocal reflected light microscopy. This study demonstrates the feasibility of creating cell-formed structural alignment combined with decellularization technique to establish decellularized anisotropic engineered tissue, which can be used to evaluate the effects of biophysical cues on stem cell or stem cell-derived cardiomyocyte differentiation and function in vitro.