The compliance of the extracellular matrix (ECM) regulates osteogenic differentiation by modulating extracellular signal-regulated kinase (ERK) activity. However, the molecular mechanism linking ECM compliance to the ERK-mitogen-activated protein kinase (MAPK) pathway remains unclear. Furthermore, RhoA has been widely implicated in integrin-mediated signaling and mechanotransduction. We studied the relationship between RhoA and ERK-MAPK signaling to determine their roles in the regulation of osteogenesis by ECM compliance. Inhibition of RhoA and ROCK in MC3T3-E1 pre-osteoblasts cultured on substrates of varying compliance reduced ERK activity, whereas constitutively active RhoA enhanced it. The expression of RUNX2, a potent osteogenic transcription factor, was increased on stiffer matrices and correlated with elevated ERK activity. Inhibition of RhoA, ROCK, or the MAPK pathway diminished RUNX2 activity and delayed the onset of osteogenesis as shown by altered osteocalcin (OCN) and bone sialoprotein (BSP) gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization. These data establish that one possible mechanism by which ECM rigidity regulates osteogenic differentiation involves MAPK activation downstream of the RhoA-ROCK signaling pathway.