Background Puberty is a sensitive period of life for developing specific skills and knowledge and acquiring abilities and attributes that are essential for managing emotions and assuming adult roles. Thus, this has implications for health educational programs to responses to the experimentation and exploration that takes place during puberty. Objectives This study aimed to examine the effect of life skills training using health literacy strategies on self-esteem and self-efficacy in adolescent female students. Methods This was a semi-experimental study with intervention-control groups performed on 96 female students (7th grade of high school) in Mashhad, Iran from January 2017 to Jun 2017. The sampling method was a multi-stage random sampling. The samples were divided into two groups: an intervention group and a control group (each one having 50 subjects) through the simple randomized approach. Data collection tools used were the Coopersmith Self-esteem Questionnaire and Sherer's Self-efficacy Questionnaire. Life skills training for the intervention group consisted of five sessions of theoretical training and workshops with role play and were designed based on health literacy strategies. The pretest, posttest and follow-up (3 months after the intervention) was conducted for both groups. Data analysis was performed using SPSS 19 software (t-test, Mann-Whitney, chi-square (χ2) and repeated data analysis). Results Before the intervention, there was no significant difference between the two groups in terms of self-esteem score (p = 0.70) and self-efficacy (p = 0.10), but immediately after training, as well as 3 months later, a significant difference was found between the two groups (p < 0.001). Conclusion Based on the findings, life skills educational interventions based on health literacy strategies could promote the self-esteem and self-efficacy abilities among female students during puberty.