Objective To study the relationship between neonatal serum 25-hydroxyvitamin D [25 (OH) D] levels and neonatal jaundice. Methods A total of 219 neonates with jaundice (experimental group) were collected, including physiological jaundice (95 cases) and pathological jaundice (124 cases). The study components were divided into three groups according to the total bilirubin (TBIL) level, including mild group (108 cases), moderate group (79 cases) and severe group (32 cases). Additionally, 100 healthy counterpart newborns were selected as the control group. Biochemical indicators and 25(OH) D levels were compared. Results The level of 25 (OH) D in the jaundiced neonatal group was significantly lower than that in healthy newborns [12.97 (7.96, 17.71) VS. 18.57 (6.95, 29.24) ng/ml, P??0.05]; 25 (OH) D level in newborn with severe jaundice [11.02 (7.53, 13.82) ng/ml] had no statistical difference with 25(OH) D levels in newborn with moderate jaundice [13.02 (8.92, 17.71) ng/ml, P?>?0.05] and children with mild jaundice (n?=?108) 25 (OH) D levels [13.50 (7.52, 19.32) ng/ml, P??0.05). The level of 25 (OH) D in the experimental group was negatively correlated with the level of total bilirubin (r?=?-0.338, P?=?0.000), and the linear regression equation was Y^?=?-2.851 X + 173.6; the level of 25 (OH) D in the experimental group was negatively correlated with the level of indirect bilirubin (r?=?-0.343, P?=?0.000), and the linear regression equation was Y^?= -2.776 X + 159.9. Conclusion 25 (OH) D may be involved in the development of neonatal jaundice. Vitamin D supplementation may help the newborns of jaundice to get the best outcome.