This aim of this current research was a multi-level analysis of the relationship between school culture, basic psychological needs, and adolescents' academic alienation. One thousand twenty-nine (N = 1,029) high school students from Qom City were randomly selected through a multi-phase cluster sampling method and answered questions regarding academic alienation, school culture, and basic psychological needs. Using HLM software for data analysis, the findings revealed that individual-level basic psychological needs had a negative and significant effect on academic alienation. Regarding school level, the effects of student--teacher relationship and educational opportunity on the academic alienation were negative and significant. In general, research findings emphasized the importance of studying multi-level factor roles and their relationship to academic alienation.