Abstract: Objective To standardize animal model of overload lactation in mice and to develop an efficient,practical method for the assessment of promoting milk secretion function.Methods Specific pathogen-free(SPF) pregnant Kunming mice were used.The numbers of neonatal mice of the first and the second time pregnant mice were recorded.The differences in body weight of second time neonatal mice before and after lactation were recorded.Survival condition of neonatal mice,the body weight of lactating mice and their offsprings were observed at different lactation time.Results The median of the neonatal mice of the first time pregnant mice(3-15 mice/nest) was 10 mice/nest,and that of the second pregnancy(5-19 mice/nest) was 14 mice/nest.The survival rate of neonatal mice in 16 newborn mice/nest group decreased statistically(P < 0.05) compared to other mice at 12th lactation day.The percentage of the nest with designed number of neonatal mice at the end of experiment was 28.6% for 14 neonatal mice/nest group and 11.1% for 16 neonatal mice/nest group.The body weight of lactating mice was significantly lowin 8 neonatal mice/nest group compared to those in other groups at the 3rd,6th,9th,18th,and 21st lactation days(P < 0.05).The increase of neonatal mice body weight decreased significantly in 14 neonatal mice/nest group compared to that of 12 neonatal mice/nest group at all lactation days(P < 0.05).There were significant decreases in accumulative milk secretion for the lactating mice of 10 and 12 neonatal mice/nest at 3rd lactation day compared to that at the 11th,14th,and 17th lactation day.Conclusion Twelve newborn mice per nest is the number of offspring mice for overloading lactation mice model in the assessment of promoting milk secretion function of health food,and the second time pregnant mice is recommended as the research objects.