Objective To analyze the reasonable and suitable level of serum 25 hydroxy vitamin D [25 (OH) D] and Vitamin D(Vit D) supplement of premature infants born less than 32 weeks in the neo-natal intensive care unit. Methods For eligible premature infants hospitalized in our department from March 2016 to December 2017,Vit D 900 IU/d was supplemented one week after birth under the conditions of es-tablishing enteral feeding. The selected cases were divided into two groups based on different blood concentra-tion of serum 25(OH)D at four weeks after birth,for 38 cases≥25 ng/ml as group A and 24 cases 15 to 25 ng/ml as group B. Their bone mass density( BMD) were tested at correct gestational age of 40 weeks and compared with 40 term infants as control group at the same period. Results The serum concentrations of 25(OH) D in group A were (29.23 ±3.08)ng/ml at 4 weeks and (35.13 ±4.67)ng/ml at 8 weeks after birth respectively. At correct gestational age of 40 weeks,13. 2％(5/38) cases demonstrated the lower BMD. The serum concentrations of 25(OH) D in group B were (20. 12 ± 3. 95)ng/ml at 4 weeks and (22. 36 ± 4. 82)ng/ml at 8 weeks after birth respectively. At correct gestational age of 40 weeks,75. 0％(18/24) cases demonstrated the lower BMD. The differences between group A and control group were not statistically sig-nificant(χ2 =0. 06,P>0. 05),and differences between group B and control group were statistically signifi-cant(χ2 =25. 45,P<0. 001). Conclusion Premature should be given Vit D 900 IU/day or more with rea-sonable and sufficient calcium and phosphorus to maintain their concentration of serum 25(OH)D at about 29. 23 ng/ml and re-check their concentration of serum 25 ( OH) D every four weeks.