Whether the maternal vitamin D deficiency is associated with infant birth weight is still an argument. Here, we performed a nested case-control study (545 women who subsequently delivered infant with macrosomia and 1090 controls) to evaluate the association of the maternal serum 25-hydroxyvitamin D [25(OH)D] concentrations with risk of macrosomia. We measured the serum 25(OH)D concentrations by enzyme immunoassays. Logistic regression analysis, receiver-operator characteristic curve analysis and graphical nomogram were used for the statistical analyses. Among women who delivered infant with macrosomia, 71.2% of the women had serum 25(OH)D concentrations <50.0 nmol/L compared with 61.1% of the control women (P < 0.001). For women with concentrations <50.0 nmol/L, they had a 33% increased risk of macrosomia compared with women whose 25(OH)D ranged from 50.0 to 74.9 nmol/L. The risk of macrosomia was significantly increased with the decreasing concentrations of serum 25(OH)D in a dose-dependent manner (P for trend = 0.001). We also observed a threshold for 25(OH)D of 50.0 nmol/L for delivering infant with macrosomia and a predictive accuracy of the 25(OH)D concentrations included panel, with an area under the ROC curve of 0.712 for delivering infant with macrosomia. In conclusion, maternal serum 25(OH)D <50.0 nmol/L is associated with delivering a macrosomic infant, and vitamin D deficiency should be monitored in pregnant women.