The present study aimed to investigate the effect of Parent-Child Interaction (PCIT) Therapy of mothers on children with separation anxiety disorder, on reducing over protection, parenting anxiety and anxiety symptoms of their 3-6 years old children. The research design was quasi-experimental with pre-test and post-test and control groups. To do so, 30 mothers of children with SAD (separation anxiety disorder) were selected using at-hand sampling and, then, they were replaced into two experimental and control groups after being matched. The research tools were included CSI-4 questionnaire (Sprafikin & Gadow, 1994), MCRE questionnaire (ROBERT, 1961), and State-trait Anxiety Inventory (STAI) (Spielberger, 1960). The parental behavior training was performed in ten 90-minute sessions in experimental group. Data were analyzed using analysis of covariance, gain score, and T-test. The results show the effectiveness of experimental treatment on reduced scores of anxiety in children of mothers in the experimental group compared with the control group. The results also showed decrease in over protection and anxiety in mothers in the experimental group. Generally speaking, the findings demonstrated that parent-child interaction education causes to reduce the separation anxiety disorder signs, parental anxiety symptoms, and over protection. Moreover, it constructs secure attachment between child and parents.