This investigation determined the contents of the fat-soluble vitamins A, D and E in human milk samples collected from Hohhot, the capital of North China’s Inner Mongolia region, and analyzed the changing patterns of these vitamins during the lactation period. Nutritional evaluation of maternal breast milk with respect to these fat-soluble vitamins was conducted by the Chinese dietary reference intake (DRI) with the aim of guiding the development of milk powder products for infants. The results showed that the contents of VA and VE were 0.11 and 0.93 mg/100 mL in colostrum, and 0.05 and 0.29 mg/100 mL in mature milk, respectively, while VD content was too low to be detected in colostrum and mature milk. VA and VE contents tended to decline with prolonged lactation. Moreover, it was found that VA was relatively abundant in breast milk from Hohhot, which could meet infants’ nutritional requirements. However, breast milk could not meet VD and VE requirements for infants. As a result, dietary fortification and supplementation of VD and VE are needed for breastfed infants.