Bone loss or insufficiency remains a great challenge for implant integrated and subsequently functional loading, where developing biomaterials to augment bone quantity and regenerate alveolar bone defects at implant site is vitally necessary. Recently, MXene, as a large new family of 2D materials, exhibits a great prospect in biomedical applications owing to its ultrathin structure and morphology with a range of extraordinary properties such as chemical, electronic, optical and biological properties etc. Besides, hydroxyapatite is a favorable biomaterial with outstanding bioactivity and osteogenic capacity. In this study, we prepared free standing UHAPNWs/MXene nanocomposite membranes via introducing ultralong hydroxyapatite nanowires (UHAPNWs) with different weight ratios into MXene to explore their potential in bone regeneration. SEM, XPS, FTIR, XRD, tensile strength, Young's modulus and water contact angles were used to characterize the morphology, chemical composition, surface properties, mechanical properties and hydrophilicity of the materials. Subsequently, in vitro studies like cell adhesion, proliferation and osteogenic differentiation of MC3T3-E1 were evaluated. The incorporation of UHAPNWs improved mechanical properties and hydrophilicity with an enhancement in cell adhesion, proliferation, and osteogenic differentiation. More importantly, 10 wt% UHAPNWs/MXene exhibited the optimal mechanical properties while biological improvement was more pronounced along with the addition of UHAPNWs when the weight fraction of UHAPNWs was from 0 to 30 wt%. Furthermore, in vivo experiments the UHAPNWs/MXene nanocomposite membranes effectively enhanced bone tissue formation quantitatively and qualitatively in a rat calvarial bone defect. Therefore, an appropriate amount of UHAPNWs into MXene plays a positive and evident role in enhancing mechanical properties, biocompatibility and osteoinductivity, leading a novel inorganic composite material for regeneration of bone tissue.