Objective To understand sexual knowledge level and acquisition situation in pupils,and provide evidence for sexuality education in primary school. Method Computer-assisted anonymous questionnaire was used among 773 pupils aged 6 to 14.Results Boys knew less about physiological knowledge compared with girls,77.9% and 74.5% of boys aged 9 to 14 didn't know nocturnal emission or menstruation was one of the main changes in male and female adolescents.48% girls aged 9 to 14 didn't know menstruation was one of the main changes in female adolescents.Pupils knew they should protect themselves,but both boys and girls knew little about the opposite sex,situation was more severer for boys (P<0.05).Of all the physiological and protection knowledge,rural ones knew less compared with city ones.Parents were the primary source and mother played a more important role in both city and rural areas.Knowledge of puberty were not concerned.Conclusion More attention should be paid on parents' role,boys and those in rural areas.Importance to menstruation and nocturnal emission should be attached to strengthen self protection consciousness.