Pueraria, the root of Pueraria lobata, is a commonly used herb in alternative medicine. This study evaluates the effect of puerarin and autogenous graft material combination on bone regeneration in calvarial critical-sized bone defects. Twenty-four rats were randomly divided into 3 groups of 8 rats each. A 5-mm diameter critical-sized defect was created in the calvarium of each animal. In group C, defects were left unfilled and were allowed to heal spontaneously without the use of any grafting material. Critical-sized bone defect created in animals receiving no treatment. In group ABG, defects were filled with autologous bone graft only. In group P-ABG, defects were filled with autologous bone graft and puerarin combination. All animals were euthanized at 28 days postoperative. Stereologic analyses were performed. New bone area and connective tissue volumes were measured. Stereologic analysis showed that the difference between grafted groups (P-ABG and ABG) and group C was statistically significant with a mean bone formation of 1.13 ± 0.09, 1.11 ± 0.11, and 0.31 ± 0.09 mm respectively (P ≤0.05). The connective tissue volumes were also statistically higher in groups P-ABG and ABG (P ≤0.05). Puerarin has positive effect on new bone formation in autogenous grafted critical-sized bone defects.