Prostheses and implants have been widely utilized in the orthopedic and dental fields. Nowadays, significant advances have been made in the structural and functional connection between living bone and prostheses, especially in the presence of compromised bone quantity/quality. Despite improvement in the treatment outcomes after augmentation, there are still challenges to meet the clinical demands due to limited materials available. In the current study, we investigated the effects of nano-nacre particles as an alternative material on stimulating bone cell differentiation and formation. Mouse osteoblastic cells (MC3T3-E1) were cultured on nano-nacre/type I collagen composite scaffold (NN-ICS) and type I collagen scaffold (ICS). Generated nano-nacre particles showed controlled release of protein and calcium for a period of 36 days. NN-ICS significantly contributed to the proliferation and differentiation of preosteoblasts compared to ICS controls. Our data showed that nano-nacre particles could serve as a candidate of bone substitution material, which potentially contributed to treatment outcomes in cases with compromised bone quality and/or quality.