Reconstruction of bone defects is still challenging for the clinician, owing to the little achievement in the effect of bone materials on osteoblastic cells. In this work, the size effect of silica nanoparticles (SNs) on cellular uptake, cytotoxicity, and cell function in the osteoblast cell line, MC3T3-E1, is studied to reveal the potentials of SNs for bone regeneration. The SNs with three different sizes are prepared using the Stöber approach and labeled with FITC. Confocal laser scanning microscopy, fluorescence-activated cell sorting and fluorescence spectrophotometry are used to evaluate the effects. All three different sized FITC-labeled silica nanoparticles have similar cellular uptake (>90%), determined by fluorescence-activated cell sorting (FACS) analysis, which suggest that all three silica nanoparticles generally have good cellular affinity. Fluorescence spectrophotometry results, however, indicate that cellular uptake is increased with a decrease in the size of the SN. Interestingly, the smaller silica nanoparticles could induce more MC3T3-E1 cell apoptosis than that of larger silica nanoparticles, and it is dose-dependent. MTT assays demonstrated that all three SNs are capable to decrease cell proliferation at a higher concentration (100 μg ml⁻¹). These results indicate that the SNs are cytotoxic, which is size and concentration-dependent. Importantly, all three SNs can directly stimulate mineralized nodule formation, which is also size-dependent. These results suggest that SNs are potentially applicable in bone regeneration, and it is possible to decrease unwanted side effects by controlling dose and size of SNs.