Objective: This study aimed to examine the sequential mediating effects of maladaptive cognitive emotion regulation strategies and separation anxiety in the relationship between harm-avoidance temperament and overprotective parenting attitude in mothers of adolescent children. Methods: A survey was administered to 210 mothers having children in the 5th and 6th grades of elementary school. Pearson correlation coefficient was determined, and the mediating effect was confirmed using Model 6 of Process Macro. Results: Maternal overprotective parenting attitude was found to be positively correlated with harm avoidance temperament, maladaptive cognitive emotion regulation strategies, and separation anxiety. In the relationship between maternal harm avoidance temperament and overprotective parenting attitude, the sequential mediating effects of maladaptive cognitive emotion regulation strategies and separation anxiety were significant, and the direct effect of maternal harm avoidance temperament on overprotective parenting attitude was also significant. Conclusions: This study provides a framework for an integrated understanding of the individual characteristics of mothers of adolescent children who display overprotective parenting attitudes by revealing the sequential mediating effects of maladaptive cognitive emotion regulation strategies and separation anxiety in the relationship between harm avoidance temperament and overprotective parenting attitudes.