The generation of titanium foams is a promising strategy for modifying the mechanical properties of intervertebral reinforcements. Thus, the aim of this study was to compare the in vitro biological response of Ti6Al4V alloys with different pore sizes for use in intervertebral implants in terms of the adhesion, proliferation, and differentiation of pre-osteoblastic cells. We studied the production of Ti6Al4V foams by powder metallurgy and the biological responses to Ti6Al4V foams were assessed in terms of different pore interconnectivities and elastic moduli. The Ti6Al4V foams obtained had similar porosities of approximately 34%, but different pore sizes (66 µm for fine Ti6Al4V and 147 µm for coarse Ti6Al4V) due to the sizes of the microsphere used. The Ti6Al4V foams had a slightly higher Young׳s modulus compared with cancellous bone. The dynamic mechanical properties of the Ti6Al4V foams were slightly low, but these materials can satisfy the requirements for intervertebral prosthesis applications. The cultured cells colonized both sizes of microspheres near the pore spaces, where they occupied almost the entire area of the microspheres when the final cell culture time was reached. No statistical differences in cell proliferation were observed; however, the cells filled the pores on fine Ti6Al4V foams but they only colonized the superficial microspheres, whereas the cells did not fill the pores on coarse Ti6Al4V foams but they were distributed throughout most of the material. In addition, the microspheres with wide pores (coarse Ti6Al4V) stimulated higher osteoblast differentiation, as demonstrated by the Alcaline Phosphatase (ALP) activity. Our in vitro results suggest that foams with wide pore facilitate internal cell colonization and stimulate osteoblast differentiation.