Purpose: The onset of substance use mostly occurs during adolescence. The aim of the present study is to investigate the relevance of personality on the basis of the NEO-Five-Factor Inventory (NEO-FFI) to future experiences with tobacco, alcohol and cannabis. Methods: The test data were derived from the baseline assessment and first follow-up of the IMAGEN study, a European multicenter and multidisciplinary research project on adolescent mental health. In the present study 1004 participants were tested. The characterization of personality was conducted with the NEO-FFI at the age of 14 (T1). The data on substance use were collected with the European School Survey Project on Alcohol and Other Drugs (ESPAD) questionnaire at the age of 16 (T2). For the statistical analysis, t-tests and univariate analyses of variance were performed. Results: The scores of Conscientiousness at T1 were significantly lower for adolescents with tobacco, alcohol and cannabis experiences at T2. We found lower scores of Agreeableness at T1 in participants with tobacco and cannabis use at T2. Extraversion at T1 was significantly higher for adolescents with smoking experiences at T2. No significant associations between Neuroticism or Openness and future substance use were observed. Conclusion: Low scores of Conscientiousness and Agreeableness seem to have the greatest value for a prediction of later experiences with substance use. As the present study is the first one to examine the predictive value of the NEO-FFI for future substance use in an adolescent sample, further studies are necessary to enable a better applicability in a clinical context.