Herein, a series of hydroxyapatite (HAp)/zein biocomposite membranes, with different inorganic/organic weight ratios, are fabricated by solvent casting method by using ultra-long hydroxyapatite nanowires as the reinforcement. The FTIR spectra and XRD analysis of HAp/zein composite membrane confirm the structure of HAp nanowires and zein in composite membranes. When the HAp nanowires content increased from 50 to 90wt%, the morphology of HAp/zein composite membrane transformed from the rough plane into the fibrous and porous structure, both Young's modulus and tensile strength exhibited about 40% of the increase, and the water absorption showed about 80% of improvement. Moreover, higher content of HAp nanowires improved the adhesion, proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells according to the examination of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, alkaline phosphatase activity, calcium deposition and gene expression. The prepared HAp/zein 9/1 composite membrane is shown as a promising candidate for the bone repair and regeneration applications.