A hierarchically structured scaffold is designed and fabricated for facilitating tendon-to-bone repair. The scaffold is composed of three regions with distinct functions: (i) an array of channels to guide the in-growth of cells and aligned deposition of collagen fibers, as well as integration of the scaffold with the tendon side, (ii) a region with a gradient in mineral composition to facilitate stress transfer between tendon and bone, and (iii) a mineralized inverse opal region to promote the integration of the scaffold with the underlying bone. Cell culture experiments confirm that adipose-derived stromal cells are able to infiltrate and proliferate through the entire thickness of the scaffold without compromised cell viability. The seeded stem cells exhibit directed differentiation into tenocytes and osteoblasts along the mineral gradient as a response to the gradient in Young's modulus. This novel scaffold holds great promise to promote the formation of a functional tendon-to-bone attachment by offering a structurally and compositionally appropriate microenvironment for healing.