Breastfeeding saves the lives of more than half a million infants in a year and cause strong emotional relationship between mother and child and their psychosocial development of personality. The aim of this study is to evaluate the effect of exclusive breastfeeding education on mothers' breastfeeding self-efficacy and stress. This experimental study with clinical trial has randomly selected three hospitals from hospitals affiliated to medical sciences universities of Tehran with intensive care units for premature infants, and 100 eligible nulliparous mothers were sampled during three months. Mothers are randomly classified into case and control groups (each group with 50 samples). The case group received breastfeeding education and educational booklet, but control group received no education. A month later, the samples reanswered to questionnaires. Data is collected through questionnaires and analyzed by descriptive and inferential statistics, T-test, paired-t, and Chi-Square tests. The results indicate that there is a significant difference between breastfeeding self-efficacy in pre and posteducational case group, so that education has significant effect on breastfeeding self-efficacy (t=10.7, p<0.01). Breastfeeding education especially in premature infants increases the mothers' breastfeeding self-efficacy, and thus the mothers with premature infants require special breastfeeding education.