Abstract: Objective To optimize the method for embedding multiple undecalcified mouse tibias in plastic blocks, improve the efficiency and stability of plastic embedding and reduce the detachment rate of plastic slides. Methods Thirty undecalcified tibias from 15 B6 mice were used for plastic embedding after calcein labeling, fixation, dehydration and infiltration. The tibias were embedded in cylindrical plastic blocks with a diameter of 4 mm. For each bone, the 1/4 proximal tibia was cut off, and the remaining 3/4 was used for re-embedding. Five bones were embedded in a single block with each bone standing closely on the surface of a flat plate. The samples were randomized into control and experimental groups in all the processes of embedding, sectioning and staining. In the 3 groups with modified embedment, flowing CO2 was added into the embedding solution, embedding solution was applied to the section surface, and the slides were heated at 95 ℃ for 15 min. The polymerization time, slide detachment rate, bone formation and osteoblast parameters were analyzed. Results We prepared 6 plastic blocks, each containing 5 tibias, whose cross sections were on the same plane. The blocks were completely polymerized and suitable for sectioning. Flowing CO2 into the embedding solution reduced the polymerization time and increased the rate of complete polymerization. Application of the embedding solution on the section surface significantly reduced the detachment rate of the sections (P<0.05) without affecting bone formation analysis (P>0.05). Heating the slides significantly lowered the detachment rate of the sections (P<0.05) without affecting osteoblast analysis (P>0.05). Conclusion The optimized method allows effective embedding of multiple undecalcified mice tibias in the same block and can be an ideal method for histological analysis of undecalcified bones.