Fluorine, a highly reactive element naturally present in air, water, and food, is mainly ingested through drinking water. The American Academy of Pediatrics recommends a daily fluoride intake of 0.05–0.07 mg/kg body weight for dental health. Excessive fluoride exposure, however, may disrupt calcium balance and reduce vitamin D levels, which are essential for maternal health during pregnancy. High fluoride levels in drinking water can alter calcium transport and reduce vitamin D levels, potentially impacting maternal health by disrupting calcium homeostasis and vitamin D metabolism. The primary objectives were to assess fluoride concentrations in drinking water, serum, and urine samples, and to examine the relationship between fluoride exposure and serum calcium and vitamin D levels among pregnant women nearing delivery. Methodology: A cross sectional, comparative study was designed, enrolling 180 pregnant women aged 18–45 with no prior history of hypercalcemia, renal conditions, or significant pregnancy complications. Participants were recruited from tertiary care hospitals in Karachi (high fluoride) and Faisalabad (low fluoride). Blood and urine samples were collected approximately one month before delivery to measure serum fluoride, calcium, and vitamin D levels. Water samples from both regions were collected and analyzed to determine fluoride concentrations, with values exceeding Finding: The results revealed that pregnant women in the high-fluoride group (Karachi) had significantly lower mean serum calcium (8.8 ± 0.5 mg/dL) and vitamin D (21.3 ± 4.2 ng/mL) levels compared to those in the low-fluoride group (Faisalabad), where mean calcium and vitamin D levels were 9.6 ± 0.4 mg/dL and 27.6 ± 3.8 ng/mL, respectively (p < 0.001). This inverse association between fluoride concentration and calcium/vitamin D levels persisted despite similar dietary and supplementation patterns between groups. Socio demographic factors and lifestyle variables were also examined,