Decellularized reproductive tissues have been used to generate biomaterials for several applications, not restricted to reproduction due to their enriched ECM and capacity to be modulated and applied for other tissues. This study aimed to produce and characterize grafts derived from decellularized uterine tissue to be used in tissue engineering approaches. Porcine uterine fragments (n=10) were decellularized in 1% SDS and 0.5% Triton X-100, followed by three cycles of ultrasonic bath. To evaluate the decellularization efficiency, HE and DAPI staining and total DNA quantification were performed. Histological analysis of ECM components was performed as well. SEM was used for ultrastructural characterization. For biomechanical characterization, native and decellularized samples were attached to a computerized mechanical testing machine and submitted to a traction charge. FTIR-ATR and Raman spectroscopy were used to perform a physical–chemical evaluation of ECM. For the cytocompatibility assay, 3T3 and canine yolk-sac-derived cells were cultured on the scaffolds for 10 days. DAPI and HE staining revealed absence of nuclei in decellularized samples; moreover, DNA quantification revealed a decrease of 95%. Regarding ultrastructure, 3D structure was maintained, conserving the original stratification and preserving thin and dense collagen bundles. Histological analyses showed that main ECM components remained preserved with a similar organization as found in the native tissue. Biomechanical results demonstrated significate difference only for the maximum pulling force between the groups, but there was no difference for maximum elongation and stiffness. Spectroscopic results also corroborated the structural findings, with no difference in the main analyzed band between the samples. In vitro assays revealed that cells were able to attach to the scaffolds, which allowed their survival and proliferation. Our data revealed that the decellularization was efficient, which preserved 3D structure, composition and biomechanical properties and presented satisfactory cytocompatibility, demonstrating the generated biomaterial can be used tissue-engineering applications.