Abstract In a previous study we demonstrated that MG-63 cells cultured on Ti-6Al-4V discs covered by alumina ceramic and submitted to intermittent mechanical strain (IMS) presented morphological alteration associated with enhanced differentiation. Here we examine how the mechanical response of osteoblasts can be modulated by the nature of the substrate. MG-63 cells were cultured on four materials: polystyrene and Ti-6Al-4V (average roughness =0.48 μm) as smooth substrates; Ti-6Al-4V (average roughness =5.76 μm) and Ti-6Al-4V covered with alumina (average roughness =5.21 μm) as rough substrates. Mechanical strains were applied for 15 min, three times a day for 1–5 days with a 600 μstrains magnitude and a 0.25 Hz frequency. IMS stimulated alkaline phosphatase activity by 25–35% on all substrates and had no effect on cell growth on either substrate. Fibronectin (FN) was chosen as representative of cell-matrix interaction. FN production was increased by 60% after 1 day of stretching only on alumina-coated discs. FN organization examined on smooth substrates was affected by 5 days of IMS, showing a thickening of the fibres. The same modifications induced by IMS were previously observed on alumina-covered discs. Vinculin expression was not affected by IMS whatever the substrate. Cell–cell interactions were determined by N-cadherin immunoblotting. N-cadherin expression was increased by IMS specifically on rough substrates. Our results suggest that the nature of the surface did not influence the up-regulation of alkaline phosphatase activity induced by IMS, but modulates specifically cell–substrate as well as cell–cell interactions in response to IMS.