This study aimed to compare the tobacco-related knowledge, attitudes and smoking behavior among Chinese medical and non-medical students across three grades from freshmen to juniors. Survey data were collected among 8,138 students using a paper-and-pencil questionnaire. Study findings indicate that compared to non-medical students, medical students in the sophomore and junior years reported significantly higher levels of knowledge regarding tobacco toxicants and tobacco-related diseases, and had stronger attitudes against smoking as personal rights and stronger attitudes in favor of smoking ban. The differences between medical and non-medical students remained after controlling for a number of covariates. However the prevalence rates of cigarette smoking were similar between medical and non-medical students across grades even after adjusted smoking initiation before entering college. Despite increases in tobacco related knowledge, approximately 40% of junior-year medical students did not recognize carbon monoxide as a toxicant from tobacco, and 30-40% of them were unknown of tobacco smoking as a risk factor for several diseases, including hypertension, stroke, and gastric ulcer. Findings of this study suggest the need for immediate action to enhance tobacco-related education in formal medical training in China to prepare future doctors for smoking prevention and to assist millions of smokers to quit.