Objectives To explore the effect of holder pasteurization, frozen storage time and thawing methods on macronutrients and energy content of donor human milk, and to provide theoretical basis for the rational use of breast milk. Methods Thirty-three samples of donor human milk were collected and an aliquot of each sample was analyzed before and after pasteurization. The remaining milk after pasteurization was split into 9 aliquot , and frozen at -20 ℃. After 30, 60, and 90 days, the milk was thawed by three different methods of room tempe-rature, 4 ℃ refrigeration, and 37 ℃ water bath, respectively. The nutrient components of each aliquot were analyzed and compared. Results We observed a mild reduction in fat and energy content after pasteurization (P <0.05). A significant decrease of fat, protein and energy content with the prolonged storage time was observed (P <0.01), and during the whole process (pasteurization + frozen storage), the decrease of fat, protein and energy content was 36.6%, 32.6%and 22.6%, respectively. The protein was influenced mostly by different thawing methods and the content of protein reached highest while thawed at 4 ℃ refrigeration. Conclusions Holder pasteurization and frozen storage at-20℃significantly reduce fat, protein and energy content of donor human milk. The donor milk should be used as quickly as possible when applied for preterm infants and thawing at 4 ℃ refrigeration is recommended before delivery to newborn infants.