Surface roughness of titanium-based implants may enhance osteogenic differentiation of cells in vitro and bone-to-implant contact in vivo. Nevertheless, how surface roughness regulates the signaling pathway of osteoblasts is little understood. The study intended to investigate specifically the roles of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in regulating osteogenic differentiation of MC3T3-E1 murine preosteoblast cells on Ti surfaces. Substrates applied were two groups of titanium disks: (1) sand-blasted and acid-etched rough surfaces (SLA) and (2) smooth pretreated Ti surfaces (PT). Surface morphology of the two groups was examined by scanning electron microscope, and cell morphology cultured on Ti disks was observed by confocal microscope. The levels of alkaline phosphatase (ALP) activity and calcium deposition were measured and compared between the two groups. Real-time polymerase chain reaction was applied to detect the expression levels of osteogenic genes including runt related protein 2 (Runx2), osterix (OSX), osteocalcin (OCN) and osteoprotegerin (OPN) of the cells cultured on the two groups of substrates and on SLA surfaces treated with ERK1/2 inhibitor, PD98095. ERK1/2 activities in MC3T3-T1 cells were measured by Western-blotting on the two surfaces with or without PD98095. Cells cultured on rougher SLA surfaces displayed a more differentiated morphology. ALP activities at 7 days and 14 days and the calcium deposition at 28 days were significantly higher on SLA surfaces. The expression levels of Runx2, OSX, OPN and OCN were upregulated by the effect of surface roughness and PD98095 further upregulated the expression levels of these osteogenic genes on SLA surfaces. ERK1/2 phosphorylation was continuously inhibited by surface roughness at 2 days, 4 days and 6 days. In contrast, no marked alterations in ERK1/2 phosphorylation on PT surfaces were observed. PT surfaces treated with PD98095 (50 μM) and SLA surfaces without PD98095 both demonstrated reduced ERK1/2 phosphorylation of the cells, and the inhibitive effect of SLA surfaces was milder than that of PD98095. In conclusion, ERK1/2 pathway may be a negative regulator of cell differentiation in a dosage-dependent manner, and the enhancing effect of surface roughness on osteoblastic differentiation may be mediated through inhibiting ERK1/2 pathway. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.