Objective To investigate the influence of anti-smoking education and environment intervention on smoking behavior of middle school students. Methods Stratified cluster random sampling method was adopted, and 9 398 middle school students were selected from 20 schools in Minhang District of Shanghai. Questionnaire survey was conducted, which was concerned with demographic characteristics, attitude and behavior towards tobacco use, school anti-smoking education, and social and family environment. One by one interview with school anti-smoking education heads was conducted, along with the evaluation of 12 school anti-smoking items such as system construction, education content, markers and environment management (6 items of smoking-free school system construction and 6 items of evaluation of anti-smoking education and environment), and the evaluation results were scored and described as good, moderate and poor. The correlation of school anti-smoking effect with smoking behavior of students and tabacco information sensitivity was analysed, and the influence of age and smoking status of parents and peers on school antismoking eduction identity was explored. Results Compared with schools with poor school anti-smoking effect, the attempted smoking rate and actual smoking rate of schools with good school anti-smoking effect decreased by 3.1% and 1.2% respectively. The school anti-smoking effect had significant influence on tobacco information sensitivity of middle school students. The identity on school anti-smoking education decreased with age, the percents of junior middle school students and senior high school students holding negative attitude towards smoking were 86.6% and 58.2% respectively, and those holding positive attitude towards smoking were 0.9% and 1.5% respectively. The recognition on harms of smoking was related to school anti-smoking education, and the identity on school anti-smoking education was related to the status of parent and peer smoking for middle school students. Conclusion School anti-smoking and environment intervention can reduce the attempted smoking rate and actual smoking rate of middle school students to some extent. However, adolescent smoking is a complicated social behavior, which requires more efficient intervention.