Wollastonite (calcium silicate) has been widely used in bone tissue engineering, but its mechanism of action on the regulation of mesenchymal stem cell proliferation and differentiation to osteoblasts still remains unclear. The current study utilized an inexpensive source of rice straw ash to synthesize wollastonite with mesoporous architecture. Mesoporous-wollastonite (m-WS) particles were characterized by transmission electron microscopy (TEM), N2 adsorption-desorption isotherms, and Fourier transform infrared (FT-IR) spectroscopy. These particles were found to be biocompatible with mouse mesenchymal stem cells (C3H10T1/2) and significantly stimulated cell proliferation by promoting the entry of the cell population from the G0/G1 phase into the S and G2/M phases via the upregulated expression of the cyclin B1 and cyclin E genes. Under osteogenic conditions, m-WS particles promoted osteoblast differentiation as indicated by calcium deposits and upregulated mRNA expression of osteoblast differentiation marker genes determined by real-time RT-PCR, depicting the osteoconductive nature of these particles. Runx2, a bone-specific transcription factor responsible for the expression of osteoblast differentiation marker genes, was upregulated in C3H10T1/2 cells. The expression of Runx2 co-regulators like Sirt-1, a positive regulator, and HDAC-4, a negative regulator, were upregulated and downregulated, respectively, by m-WS particles in these cells. Thus, this study provides a detailed insight into the effect of m-WS particles on mesenchymal stem cells at the molecular and cellular levels for in vitro bone formation.