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.