Native tissues are endowed with a highly organized nanofibrous extracellular matrix (ECM) that directs cellular distribution and function. The objective of this study is to create a purely natural, uniform, and highly aligned nanofibrous ECM scaffold for potential tissue engineering applications. Synthetic nanogratings (130 nm in depth) are used to direct the growth of human dermal fibroblasts for up to 8 weeks, resulting in a uniform 70 μm-thick fibroblast cell sheet with highly aligned cells and ECM nanofibers. A natural ECM scaffold with uniformly aligned nanofibers of 78 ± 9 nm in diameter is generated after removing the cellular components from the fibroblast sheet. The elastic modulus of the scaffold is well maintained after the decellularization process because of the preservation of elastin fibers. Reseeding human mesenchymal stem cells (hMSCs) shows the excellent capacity of the scaffold in directing and supporting cell alignment and proliferation along the underlying fibers. The scaffold's biocompatibility is further examined by an in vitro inflammation assay with seeded macrophages. The aligned ECM scaffold induces a significantly lower immune response compared to its unaligned counterpart, as detected by the pro-inflammatory cytokines secreted from macrophages. The aligned nanofibrous ECM scaffold holds great potential in engineering organized tissues.