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Introduction: Adolescence is a critical period for physical and mental development. The effect of early life stress on mood disorders has been intensively studied in adults using rodent models, but it has been less studied in adolescents. The present study aimed to examine the effect of early life stress on anxiety‐related and depression‐like behaviors in adolescent C57BL/6 mice and the sex difference. Methods: C57BL/6 mice of both sexes were used, and early life stressors included maternal separation (MS, P2‐12, 4 hr per day), restraint stress (RS, P33 to 39, 4 hr per day), and their combination (MRS). Open field test, elevated plus maze, and forced swimming test were performed at different time points during adolescence and adulthood. Results: It was found that MS did not affect the anxiety‐related behaviors of both males and females tested on P30‐31 and P41‐42. RS decreased the anxiety level in adolescent males but did not affect it in the females. MS, RS, and MRS all significantly increased the depression‐like behavior in adolescent males, but only MRS increased the depression‐like behavior in adolescent females. All effects on adolescent males and females did not persist into adulthood. Conclusion: The present results showed that early life stress affected anxiety‐related and depression‐like behavior in adolescent mice in manners depending on the nature of stress, the developmental period, and sex. [ABSTRACT FROM AUTHOR]