Objective To investigate the college students’ knowledge ,attitudes and practices concerning antibiotics use in Nanjing and to provide the evidence for formulating policy on rational use of drugs by analyzing the influencing factors . Methods A self-designed Likert scale questionnaire was used to investigate a cluster sampling of 618 students of 5 colleges. Results A total of 600 subjects completed the survey instrument, the correct rate of medication knowledge was 55.07%.Logistic regression analysis identified major, grade and home town as independent risk factors for irrational use concerning antibiotics (P<0.01). Conclusion Undergraduate students had inadequate knowledge, moderately accurate beliefs and inappropriate practices concerning antibiotics. Education on medication knowledge for college students especially for non-medical students should be strengthened, so as to change the wrong medication concept and bad habits and improve the rationality of medication practices.