Sex education is one of the most challenging topics for mothers. Therefore, we examined the effect of sexual health education of mothers on their comfort and intention to discuss sex-related topics with adolescent girls. In this controlled intervention study, 160 mothers of 13- to 16-year-old girls were randomly assigned to either the intervention or control groups. The intervention group underwent sexual health training, while the control group received routine care. Paired t-test, chi-square, analysis of covariance, and multivariate analysis of covariance were used for the analysis of data. There was a significant increase in mean parents' comfort in discussing sex topics in the intervention group compared to the control group (p < .001). The intervention group had spoken with their child to a higher extent than the control group in all 13 items of parents' Intention index for discussing sex topics with the child (except for delivery and menstruation) (p < .05).There was no significant difference between groups for the mean age appropriate for discussing sex topics with the child, Sense of Competence as well as its dimensions, such as self-efficacy and satisfaction. Mean pre-to-post scores of sense of Competence in child rearing (p = .04) and self-efficacy (p = .037) significantly increased in the intervention groups, while they were not significant in the control group. Sexual health education was effective in the feeling of interest and comfort of mothers to discuss sex topics. Therefore, counseling services on mothers' speech interactions are recommended to promote adolescent girl's sexual health.