Prolonged labor is associated with various maternal and neonatal complications. This study aims to investigate the relationship between 25-hydroxyvitamin D3 levels and pain intensity and duration of labor stages in primiparous women. This cross-sectional study was conducted in Iran from November 2021 to January 2022 and comprised primiparous women who were in active labor after a term pregnancy (37-42 weeks). Five milliliter of blood was taken from each subject and centrifuged for the measurement of vitamin D level using the enzyme-linked immunosorbent assay method. The High-Performance Liquid Chromatography (HLPC) method was used to measure 25-OH vitamin D. In addition, through history, examination, and investigations, the subjects were evaluated according to the pain intensity and duration of the first (active phase) and second stages of labor. The results of the Pearson correlation test indicated a significant relationship between vitamin D and active phase duration (r = 0.64, p = 0.012), second stage duration (r = 0.73, p = 0.001), pain intensity of the active phase (r = 0.61, p = 0.022), and pain intensity of the second stage (r = 0.65, p = 0.026). According to the analysis of variance table, based on vitamin D, there were statistically significant differences between the groups in terms of the active phase duration, second stage duration, pain intensity of the active phase, and that of the second stage of labor (p < 0.05). Low levels of vitamin D may influence the progress of labor and increase the rate of prolonged labor.