In vitro tissue engineering requires a progenitor cell source and a porous scaffold providing three dimensional (3D) supports for growth and differentiation to attain tissue architectures. This research focused on fabrication and characterization of 3D porous scaffolds using chitosan (CS), collagen (CG) and chitosan-collagen (CS-CG) composite to investigate their influence on human mesenchymal stem cell (hMSC) adhesion, proliferation and differentiation. Material dependent variations in porous morphology and mechanical behavior of the fabricated CS, CG and CS-CG scaffold showed significant impact on hMSC adhesion, proliferation and differentiation. The maximum hMSC adhesion and proliferation was reported on CS-CG scaffold among all fabricated scaffold groups. Interconnectivity of pores structure in CS-CG scaffold was considered as preferable attribute for such enhanced growth and distribution throughout the scaffold. Besides, CS scaffold with well interconnected pores showed poor adhesion and proliferation because of inadequate adhesion motifs. In case of CG scaffold, optimum growth and distribution of hMSC occurs only at the surface because of the absence of interconnectivity in their pore structures. Likewise, osteogenic differentiation of hMSC occurs most preferably in CS-CG composite scaffold among all scaffold groups. Such enhanced hMSC proliferation and differentiation in CS-CG scaffold significantly influenced on mechanical behavior of scaffold which is essential for in vivo application of a bone tissue implant. Thus CS-CG composite scaffold holds promise to be a suitable platform for in vitro engineering of bone tissue implant.