Several studies consider vitamin D deficiency as a modifiable risk factor for adverse pregnancy outcomes. However, there is no specific cut-off point for the serum level of this prohormone to identify high-risk pregnant women. This study aimed to determine the thresholds for the circulating levels of 25-hydroxyvitamin D (25(OH)D), associated with adverse pregnancy outcomes, including preterm labor, preeclampsia (PE), and gestational diabetes mellitus (GDM), using a generalized additive model. This is a descriptive and analytical cross-sectional study carried out on the data collected from the Khuzestan Vitamin D Deficiency Screening Program in Pregnancy. Of 1800 pregnant women, referred to the health centers of Masjed-Soleyman and Shushtar (Khuzestan Province, Iran), we used the data of 1763 pregnant women, whose serum vitamin D status during the third trimester of pregnancy was available. The datasets were randomly divided into training (70%) and validation (30%) subsets. The cut-off levels of 25(OH)D were identified for the low, moderate, and high risk of adverse pregnancy outcomes, according to generalized additive models (GAM) with smooth functions in the training data set. Then Generalized Linear Model (GLM), with logit link function was applied in the validation dataset to explore the relationships between the optimal vitamin D classification and adverse pregnancy outcomes after adjusting for the potential confounders. The optimal cut-off levels of 25(OH)D for the high, moderate, and low risk of GDM were ≤16, 16-26, and >26 ng/mL, respectively. Also, the optimal cut-off points of 25(OH)D for the high, moderate, and low risk of preterm delivery were ≤15, 15-21, and >21 ng/mL, respectively. Finally, the corresponding values for the high, moderate, and low risk of PE were ≤15, 15-23, and >23 ng/mL, respectively. The models were well-calibrated, based on the Hosmer-Lemeshow test. Results of the adjusted generalized linear model showed a significant increasing trend in the risk of pregnancy outcomes by decreasing 25(OH)D levels. In the preconception period, a 25(OH)D cut-off level of >15 ng/mL is recommended for the prevention of adverse pregnancy outcomes. Copyright © 2020 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.