Phosphorylated materials are attractive candidates for bone regeneration because they may facilitate the construction of a phosphorylated bone extracellular matrix (ECM) to build a beneficial environment for bone formation. Here, we designed and synthesized a new phosphorylated material, collagen type I phosphorylated with alendronate sodium (Col-Aln), based on the biodegradable osteoconductive collagen backbone. Col-Aln can distinctly accelerate in vitro mineralization in simulated body fluid. Col-Aln showed good biocompatibility with bone marrow mesenchymal stem cells (BMSCs) and promoted their adhesion as well as the osteogenic differentiation of BMSCs more effectively than did pure collagen. Furthermore, collagen and Col-Aln scaffolds implanted into a critical-sized rat cranial defect for 4 and 8 weeks were shown to degrade in vivo and helped to facilitate bone growth in the defect, while the phosphate-containing Col-Aln scaffold significantly promoted new bone formation. Col-Aln provides a new strategy to integrate bioactive phosphate molecules via covalent grafting onto biopolymers and has promise for bone regeneration applications.