Objective: The purpose of this study was to examine the direct and indirect effects (through working mothers’ overprotective parenting behavior) of working mothers’ separation anxiety and job satisfaction on preschoolers’ separation anxiety. Methods: A total of 251 working mothers with preschool-aged children participated in this study. Mothers completed a questionnaire concerning their separation anxiety, job satisfaction, overprotective parenting behavior, and preschoolers’ separation anxiety. Data were analyzed using t-test, one-way ANOVAs, partial correlations and path analysis. Results: In terms of direct effects, working mothers’ separation anxiety had a direct effect on preschoolers’ separation anxiety. However, job satisfaction did not have a direct effect on preschoolers’ separation anxiety. With respect to indirect effects, working mothers’ separation anxiety indirectly influenced preschoolers’ separation anxiety through their overprotective parenting behavior. However, mothers’ job satisfaction did not have an indirect effect on preschoolers’ separation anxiety. Conclusion/Implications: Findings from this study emphasize the importance of reducing mothers’ separation anxiety in order to prevent preschoolers’ separation anxiety.