Background: Although the frequency of twin births has increased in Japan, little information is available about breastfeeding twins. An intervention of breastfeeding support was implemented to promote breastfeeding for mothers of twins and to compare the factors related to breastfed and formula-fed twins. Methods: Eleven Japanese twins-mother pairs received breastfeeding support according to a breastfeeding protocol for twins, and a control group of seven pairs received routine care. Perinatal data were obtained from clinical records for analysis. Results: No significant differences were evident between the two groups in terms of maternal age, gestational age, birthweight, the age at which breastfeeding was initiated, amount of supplementary feeding in hospital, infants' age at discharge, or duration of mothers' hospital stay. At age 3 months, 13 infants in the intervention group were being exclusively breastfed and 9 were almost exclusively breastfed. In the control group 4 infants were almost exclusively and 10 were exclusively formula-fed. The mothers in the intervention group breastfed and expressed milk significantly more frequently than the mothers in the control group during their hospital stay. Conclusions: Mothers of twins face many obstacles in breastfeeding, and health care professionals should offer individualized care and support.