Background Tissue engineering (TE) seeks to fabricate implants that mimic the mechanical strength, structure, and composition of native tissues. Cartilage TE requires the development of functional personalized implants with cartilage-like mechanical properties capable of sustaining high loadbearing environments to integrate into the surrounding tissue of the cartilage defect. Objective In this study we evaluated the novel 1,4-Butanediol thermoplastic polyurethane elastomer (b-TPUe) derivative filament as a 3D bioprinting material with application in cartilage TE. Methods The mechanical behaviour of b-TPUe in terms of friction and elasticity were examined and compared with human articular cartilage, PCL, and PLA. Moreover, infrapatellar fat pad-derived human mesenchymal stem cells (MSCs) were bioprinted together with scaffolds. In vitro cytotoxicity, proliferative potential, cell viability, and chondrogenic differentiation were analysed by Alamar blue assay, SEM, confocal microscopy, and RT-qPCR. Moreover, in vivo biocompatibility and host integration were analysed. Results This article is protected by copyright. All rights reserved. b-TPUe demonstrated a much closer compression and shear behaviour to native cartilage than PCL and PLA, as well as closer tribological properties to cartilage. Moreover, b-TPUe bioprinted scaffolds were able to maintain proper proliferative potential, cell viability, and supported MSCs chondrogenesis. Finally, in vivo studies revealed no toxic effects 21 days after scaffolds implantation, extracellular matrix (ECM) deposition and integration within the surrounding tissue. Conclusions This is the first study that validates the biocompatibility of b-TPUe for 3D bioprinting. Our findings indicate that this biomaterial can be exploited for the automated biofabrication of artificial tissues with tailorable mechanical properties including the great potential for cartilage TE applications.