We have evaluated the in-vitro biocompatibility of Ti6Al4V alloy coated by plasma spraying with an identical alloy. These surfaces are widely used in cementless prosthetic components, although osteoblasts behavior on this treated alloy has not been evaluated to date. Cross sectional examination revealed a thick and rough coating of identical composition without sign of delamination. Within the coating, small discontinuities and nonconnected pores were observed. Osteoblast response was evaluated by assessing cell adhesion, proliferation, and differentiation of primary cultures of human osteoblastic cells. Compared to the polished alloy, osteoblast adhesion measured as cell attachment and actin network reorganization was delayed on the plasma-sprayed surface. Cell proliferation and viability were also impaired on the rough surface. Several informative markers of osteoblastic differentiation such as procollagen I peptide, alkaline phosphatase, osteocalcin, osteoprotegerin, and mineralized nodule formation were evaluated and indicated that the plasma-sprayed alloy favored a more differentiated phenotype than polished alloy. Taken together, our in vitro results indicate that successful osseointegration of plasma spraying of Ti6Al4V with an identical alloy is mediated by modulation of osteoblastic differentiation and mineralization.