Resin-based composites are used for bone repair applications and comprise resin matrix and different sized filler particles. Nanometer-sized filler particles improve composite's mechanical properties compared with micrometer-sized filler particles, but whether differences exist in the biological response to these composites is unknown. Natural bone comprises a nanocomposite structure, and nanoscale interactions with extracellular matrix components influence stem cell differentiation. Therefore we hypothesized that nanometer-sized filler particles in resin-based composites enhance osteogenic differentiation of stem cells showing a more bone cell-like response to mechanical loading compared with micrometer-sized filler particles. Pulsating fluid flow (PFF; 5 Hz, mean shear stress: 0.7 Pa; 1 h) rapidly, within 5 min, increased nitric oxide production in human adipose stem cells (hASCs) on nanocomposite, but not on micro-hybrid composite. PFF increased RUNX2 expression in hASCs on micro-hybrid composite, but not on nanocomposite after 2 h post-incubation. PFF did not affect mean cell orientation and shape index of hASCs on both composites. In conclusion, the PFF-increased nitric oxide production in hASCs on nanocomposite, and increased osteogenic differentiation of hASCs on micro-hybrid composite suggest different responses to mechanical loading of hASCs on composite with nanometer-sized and micrometer-sized filler particles. This might have important implications for bone tissue engineering.