Nacre, exhibiting excellent mechanical strengths due to its hierarchical structures, becomes a potential model to design bone implants. Herein, we firstly fabricated nacre-mimetic hydroxyapatite/chitosan/gelatin (HA/CS/Gel) layered scaffolds and incorporated substance P (SP) peptides. The CS scaffolds with a layered architecture could regulate the deposition of HA nanoplates on the interlamellar CS sheets by using CaCO3 as precursors. The HA/CS/Gel layered scaffolds exhibited a great flexure strength of 11.42 MPa due to a brick-and-mortar structure. The biocompatible components, layered macropores and SP peptides in the HA/CS/Gel therapeutic scaffolds facilitated the spreading and proliferation mesenchymal stem cells (MSCs). Notably, the incorporation of SP peptides induced MSC osteogenic differentiation and extracellular matrix mineralization. Rabbit knee subchondral defect models further proved that the HA/CS/Gel-SP layered scaffolds promoted in vivo subchondral bone regeneration. Hence, the combination of nacre-mimetic bone implants and therapeutic drugs may become an attractive strategy for subchondral bone regeneration.