Despite the importance of school climate for positive student health outcomes, there is still little research addressing its potential effects on substance use. The aim of this study was to examine whether dimensions of school climate: respectful treatment, discrimination, rules, safety and harassment relate to alcohol, tobacco and marijuana use in a sample of Chilean students. The sample comprised 182.066 year 10 students in 2.907 schools, who completed the Questionnaire of Quality and Context of Education as part of the SIMCE evaluation in 2017. Statistical analyses included Confirmatory Factor Analysis (CFA), multilevel logistic regression and the estimation of predicted probabilities. Models were adjusted for sex and school socioeconomic status. The results showed that school climate was associated with adolescent alcohol, tobacco and marijuana use. Negative treatment of students, discrimination, poorly-defined school rules, inadequate security, and perceived bullying were associated with an increased risk of substance use. School interventions addressing adolescent substance use should consider the influence of school climate on risk behaviors.