Two-dimensional (2D) nanomaterials are attracting more and more interest in regenerative medicine due to their unique properties; however 2D biomimetic calcium mineral has not yet been developed and demonstrated application for bone tissue engineering. Here we described a novel calcium phosphate material with a 2D nanostructure that was synthesized using collagen and sodium alginate as the template. In vitro performance of the nanocrystalline material was evaluated, and we found that 2D CaP nanoparticles (NPs) enhanced the in vitro osteogenic differentiation of rat mesenchymal stem cells (rMSCs) through a macrophage-mediated signal pathway, when co-cultured with RAW 264.7 cells, rather than direct NP/stem cell interaction. A 2D topology structured surface was constructed by encapsulating the CaP nanomaterials in a gelatin hydrogel, which was demonstrated to be able to mediate in vivo ossification through a macrophage polarization related pathway in a femur defect rat model, and allowed the optimal therapeutic outcome compared to normal CaP counterparts. Our current work may have enlightened a new mechanism regarding NP-induced stem cell differentiation through immunoregulation, and the 2D CaP encapsulated hydrogel scaffold may serve as a potential alternative to autograft bone for orthopedic applications.