Macroporous tantalum (Ta) coating was produced on titanium alloy implant for bone repair by cold spray (CS) technology, which is a promising technology for oxygen sensitive materials. The surface characteristics as well as in vitro cytocompatibility were systematically evaluated. The results showed that a rough and macroporous CS-Ta coating was formed on the Ti6Al4V (TC4) alloy surfaces. The surface roughness showed a significant enhancement from 17.06 μm (CS-Ta-S), 27.48 μm (CS-Ta-M) to 39.21 μm (CS-Ta-L) with the increase of the average pore diameter of CS-Ta coatings from 138.25 μm, 198.25 μm to 355.56 μm. In vitro results showed that macroporous CS-Ta structure with tantalum pentoxide (Ta2O5) was more favorable to induce human bone marrow derived mesenchymal stem cells (HBMSCs) spreading, migration and osteodifferentiation than TC4. Compared with the micro-scaled structure outside the macropores, the surface micro-nano structure inside the macropores was more favorable to promote osteodifferentiation with enhanced alkaline phosphatase (ALP) activity and extracellular matrix (ECM) mineralization. In particular, CS-Ta-L with the largest pore size showed significantly enhanced integrin-α5 expression, cell migration, ALP activity, ECM mineralization as well as osteogenic-related genes including ALP, osteopontin (OPN) and osteocalcin (OCN) expression. Our results indicated that macroporous Ta coatings by CS, especially CS-Ta-L, may be promising for hard tissue repairs.