Introduction: Vitamin D3 deficiency, along with iron and other nutrient deficiencies such as carbohydrates, proteins, vitamins, and minerals, can influence maternal and fetal health outcomes. The aim of this study was to assess Vitamin D3 levels in anemic and non-anemic rural pregnant women –A case control study. Methods:This one-year case-control study at Bhagat Phool Singh Medical College involves 400 rural patients aged 21-35. Simple random sampling is used. Participant’s medical histories, BMI, and nutritional status are assessed. Pregnant women's, serum ferritin, and vitamin D levels are measured, classifying them into anemic (<10gm/dl) and non-anemic (>10gm/dl) groups. Results: The mean ages for Group 1 and Group 2 were 24.63 ± 4.17 and 25.12 ± 3.97 years, respectively. The study found significant differences in mean Vitamin D levels between Group 1(13.42 ng/mL ± 8.94) and Group 2 (18.78 ng/mL ± 10.66) with a P value of 0.001. Vitamin D levels across different ferritin categories, significant differences were observed (P = 0.03). Participants with vitamin D deficiency (< 20 ng/mL) showed higher percentages of ferritin levels below 50 ng/mL (78.7%) compared to those with insufficient (72.1%) and normal (60.7%) vitamin D levels. Analysis of supplemental interventions showed no significant difference in the use of vitamin D tablets along with calcium supplements (P value = 0.22) or iron tablets/syrup (P value = 0.22). Vitamin D injections were administered to 7.5% of Group 1 and 11.5% of Group 2, with no statistically significant distinction (P value = 0.17). Conclusion: The primary objective of the study was to evaluate and compare serum vitamin D3 levels between anemic and non-anemic rural pregnant women, aiming to identify significant differences. Secondary objectives included assessing vitamin D3 deficiency incidence and exploring the association between vitamin D3 and serum ferritin levels, aiming to understand their impact on maternal health and anemia. The study highlights vitamin D deficiency as a potential risk factor for anemia in pregnant women, advocating for interventions to optimize maternal vitamin D levels and improve hemoglobin status Keywords: Rural pregnant women, Vitamin D, Anemia.