Objective: The purpose of this study was to compare the plasma oxytocin levels of children with separation anxiety disorder (SAD) and their mothers with those of healthy controls and to examine the relationship between oxytocin levels and changes in anxiety three months after treatment. Methods: Thirty children aged 6−12 years with SAD, 30 healthy children, and mothers of both groups were included in the study. All cases were evaluated with semi-structured interview and Clinical Global Impression Scale. All cases and mothers of both groups filled out scales to determine various psychological variables (anxiety, depression, and attachment). The patient group children were re-evaluated with their mothers after three months, following treatment. Plasma oxytocin levels were evaluated from both groups and their mothers before and after treatment. Results: The plasma oxytocin levels of mothers of children with SAD were significantly lower than those of the controls and increased significantly three months after their children were treated. No difference was found between the plasma oxytocin levels of children with SAD and the control group, and these children’s levels decreased significantly after treatment. A positive correlation was found between changes in the plasma oxytocin levels of children with SAD and the change in anxiety scores. Conclusion Our results show that the change in plasma oxytocin levels in both children and mothers after treatment suggests that oxytocin may be important in the etiology of SAD.