OBJECTIVE To study the dynamic changes in macronutrients and energy in human milk from mothers of premature infants. METHODS A total of 339 human milk samples were collected from 170 women who delivered preterm or full-term infants in the Department of Obstetrics and Gynecology, Peking Union Medical College Hospital between November 2012 and January 2014. Macronutrients (proteins, fats and carbohydrates and energy were measured using a MIRIS human milk analyzer and compared between groups. RESULTS In milk samples from premature infants' mothers, the protein levels were the highest in colostrum (2.22±0.49 g/dL), less in transitional milk (1.83±0.39 g/dL), and the least in mature milk (1.40±0.28 g/dL) (P<0.01), and the levels of fats (2.4±1.3 g/dL vs 3.1±1.1 g/dL; P<0.01), carbohydrates (6.4±0.9 g/dL vs 6.6±0.4 g/dL; P<0.05) and energy (55±9 kcal/dL vs 62±8 kcal/dL; P<0.01) were significantly lower in colostrum than in transitional milk. The protein levels in colostrum from premature infants' mothers were significantly higher than those in colostrum from term infants' mothers (2.22±0.49 g/dL vs 2.07±0.34 g/dL; P<0.05). The colostrum from mothers of premature infants with a gestational age of ≤30 weeks had significantly higher protein levels than those from mothers of premature infants with gestational ages of 30(+1)-33(+6) weeks and ≥34 weeks (2.48±0.68 g/dL vs 2.11±0.25 g/dL and 2.22±0.39 g/dL respectively, P<0.05); the energy levels in colostrum from mothers of premature infants with a gestational age of ≤30 weeks group (51±6 kcal/dL) were significantly lower than those in colostrum from mothers of premature infants with a gestational age of 30(+1)-33(+6) weeks (58±8 kcal/d; P<0.05). The carbohydrate levels in transitional milk from mothers of premature infants with a gestational age of ≤30 weeks were significantly higher than those in transitional milk from mothers of premature infants with gestational ages of 30(+1)-33(+6) weeks and ≥34 weeks (P<0.05). The protein levels in mature milk from mothers of premature infants with a gestational age of 30(+1)-33(+6) weeks were significantly higher than those in mature milk from mothers of premature infants with gestational ages of ≤30 weeks and ≥34 weeks (P<0.05). CONCLUSIONS The levels of macronutrients and energy in milk from mothers of premature infants vary significantly between colostrum, transitional milk, and mature milk. Protein levels are significantly higher in colostrum from premature infants' mothers than in colostrum from term infants' mothers, but the significant difference is not seen for mature milk. Macronutrient and energy levels show significant differences between milk samples from mothers of premature infants with different gestational ages, so as to meet different needs of premature infants.