The Hox gene plays a crucial role in the bone development, determining their structure and morphology. Limb bone grafts expressing Hox positive genes are commonly used for free transplantation to repair Hox negative mandibular critical bone defects. However, the specific role of original Hox genes in newly formed bone during the cross-layer bone grafting healing process remains unexplored. Our findings demonstrate that femurs ectopically grafted into the mandibular environment retained a significant ability to differentiate into cartilage and form cartilaginous callus, which may be a key factor contributing to differences in bone graft healing. Hoxc10, an embryonic layer-specific genes, regulates cartilage formation during bone healing. Mechanistically, we observed Hoxc10 retention in co-cultured femoral BMSCs. Knocking out Hoxc10 narrows the bone gap and reduces cartilage formation. In summary, we reveal Hoxc10's 'positional memory' after adult cross-layer bone graft, influencing the outcomes of autologous bone graft.