The myotendinous junction (MTJ) facilitates force transmission between muscle and tendon to produce joint movement. The complex microarchitecture and regional mechanical heterogeneity of the myotendinous junction pose major challenges in creating this interface . Engineering this junction is challenging due to substantial fabrication difficulties in creating scaffolds with intricate microarchitecture and stiffness heterogeneity to mimic the native muscle-tendon interface. To address the current challenges in creating the MTJ , digital light processing (DLP)-based 3D printing was used to fabricate poly(glycerol sebacate)acrylate (PGSA)-based muscle-tendon scaffolds with physiologically informed microstructure and mechanical properties. Local mechanical properties in various regions of the scaffold were tuned by adjusting the exposure time and light intensity used during the continuous DLP-based 3D printing process to match the mechanical properties present in distinct regions of native muscle-tendon tissue using printing parameters defined by an artificial intelligence-trained algorithm. To evaluate how the presence of zonal stiffness regions can affect the phenotype of a 3D-printed MTJ model, three 3D-printed PGSA-based scaffold conditions were investigated: (1) a scaffold with muscle-informed mechanical properties in its entirety without zonal stiffness regions, (2) a scaffold with one end possessing native muscle stiffness and the other end possessing native tendon stiffness, and (3) a scaffold with three distinct regions whose stiffness values correspond to those of muscle on one end of the scaffold, MTJ in the middle junction of the scaffold, and tendon on the other end of the scaffold. The scaffold containing regional mechanical heterogeneity most similar to the native MTJ (condition 3) was found to enhance the expression of MTJ-related markers compared to those without the presence of zonal stiffness regions. Overall, the DLP-based 3D printing platform and biomaterial system developed in this study could serve as a useful tool for mimicking the complexity of the native MTJ, which possesses inherent geometric and mechanical heterogeneity.