Negative peer attitudes are generally recognised as being a major barrier to full social inclusion at school for children and youth with disabilities. The present study examined the attitudes of 1,872 grade nine high school students in Ontario, Canada toward their peers with disabilities. A bioecological perspective and a structural equation modeling approach were adopted to investigate how various aspects of school culture and student interpersonal factors influenced attitudes. The majority of students (61%) held attitudes toward peers with disabilities that ranged from slightly above neutral to very positive. However, a substantial number (21%) held slightly below neutral to very negative attitudes. Positive student relationships at the school level and a school goal task structure that promoted learning and understanding for all students, rather than social comparison and competition among students, were two aspects of school culture that had both direct associations with positive attitudes and indirect associations through student interpersonal factors. Teacher and student relationships at the school level was an aspect of school culture that had an indirect association with positive attitudes via interpersonal support from teachers. Results support the development of ecologically based programs aimed at promoting aspects of school culture that contribute to positive attitudes of students toward their peers with disabilities.