Titanium (Ti) is the material of choice for dental and orthopaedic implants due to its highly biocompatible nature. Modification of the implant surface, either topographically (as roughness) or chemically, can promote accelerated osteogenesis in vivo and greatly increase bone-implant contact and bonding strength. In this paper, we sought to characterise the cellular and molecular responses of human bone marrow-derived mesenchymal stromal cells (hMSCs) to two modified Ti surfaces: a rough hydrophobic surface that was sand-blasted and acid-etched (SLA) and an SLA surface of the same roughness that was chemically modified to have high wettability/hydrophilicity (SLActive). A smooth polished (SMO) Ti surface was used as a control. Whilst no differences in initial cell attachment to any of the surfaces were observed, we found that hMSCs cultured on the rough surfaces underwent a decrease in cell number early in culture, yet simultaneously expressed higher levels of the osteogenic markers SPP1, RUNX2 and BSP. Furthermore, deposits of calcified matrix were observed at earlier time points on both SLA and SLActive surfaces compared to SMO and this correlated with increased expression of the osteogenic promoter WNT5A in response to the rough surfaces. Osteogenic responses to SLActive were moderately better than the hydrophobic SLA surface and gene expression studies indicate that WNT5A activation may be responsible for this increased osteogenic differentiation.