This study compared the protein composition of breast milk and the nutrient intake between Thai and Japanese lactating mothers. The breast milk was collected from 15 Thai and 14 Japanese mothers at the fifth day post-partum. Twenty-four-hour dietary records were performed from the second-to-the-fourth day post-partum. The nutrient intake was calculated by using the nutrient content of a food table. The protein composition of the whey was separated by gel electrophoresis and was identified by mass spectrometry and two-dimensional electrophoresis. The results showed that the concentrations of the major protein types in the breast milk were not significantly different between the two groups. The concentrations of the minor protein types varied markedly with the individuals, with higher concentrations in the breast milk of the Thai mothers. There were no significant differences in terms of the energy and protein intake; however, the sources of energy were different. The results indicate that the total protein and lactoferrin concentrations in the breast milk could be predicted by the maternal daily energy and fat intake.