Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D 3 [1 α,25(OH) 2D 3]. The observations that β 1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and that it is regulated by 1 α,25(OH) 2D 3 in a surface-dependent manner, suggest that β 1 may play a role in mediating osteoblast response. To test this hypothesis, we silenced β 1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the β 1-silenced osteoblasts to surface microtopography and 1 α,25(OH) 2D 3. To better understand the role of β 1, MG63 cells were also treated with two different monoclonal antibodies to human β 1 to block ligand binding. β 1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β1, prostaglandin E 2, and osteoprotegerin in comparison with control cells. Moreover, β 1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1 α,25(OH) 2D 3. Anti β 1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z caused dose-dependent decreases in cell number and alkaline phosphatase and an increase in osteocalcin. Effects of 1 α,25(OH) 2D 3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β 1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1 α,25(OH) 2D 3. The results also show that β 1 mediates, in part, the synergistic effects of surface roughness and 1 α,25(OH) 2D 3.