A high prevalence of vitamin D deficiency (VDD) during early pregnancy has been reported globally, along with a high risk of adverse pregnancy and birth outcomes. The present cut-off to diagnose VDD during pregnancy is <20 ng/mL of serum 25-hydroxyvitamin-D (25(OH)D) concentration, but there is a lack of consensus on this value. We evaluated this diagnostic cut-off specifically during early pregnancy among apparently healthy Indian women. Demographic details, obstetrics history, anthropometric measurements, and blood samples were collected from 395 apparently healthy pregnant Indian women at ≤14 weeks of gestation, after obtaining written informed consent. The inverse relationship between 25(OH)D and parathyroid hormone (PTH) concentrations was examined to define the breakpoint at which PTH was maximally suppressed using a segmented regression analysis. Covariate exposures associated with VDD were also examined. The breakpoint at which a sharp increase in PTH was observed in response to decreasing 25(OH)D concentrations occurred at 15.76 ng/mL (95%CI: 12.3-19.2; < 0.001). Using this diagnostic threshold, 66.1% of pregnant women were VDD compared to 82.0% when using the present cut-off. Statistically significant associations between VDD and parity ( = 0.011), season (winter: = 0.001; post-monsoon: < 0.001), anemia status ( = 0.044), and physical activity ( = 0.045) were also found. : Our diagnostic cut-off for VDD, derived from PTH regulation in early pregnancy, is lower than the currently recommended threshold. Although assessing vitamin D status may be challenging due to the influence of modifiable and non-modifiable factors such as parity, anemia, season, and physical activity. These findings underscore the need to re-evaluate existing cut-offs through well-designed longitudinal studies to prove causality between this threshold and adverse pregnancy outcomes.