Patients affected with hypothyroidism are largely characterized by disruption of metabolism and mineral levels. Also known as cholecalciferol, vitamin D3 is an important regulator of calcium and phosphate homeostasis. The study aims to assess the connection between serum vitamin D3, calcium, and phosphate levels in hypothyroid female patients aged 45-85 years in Najaf province of Iraq. A case-control study was conducted at Al-Najaf Teaching Hospital on 80 females. The study population consisted of a total of 80 patients diagnosed with hypothyroidism and 40 cases suffering from the same conditions of similar age as the sufferer. We determined serum vitamin D3 levels by electrochemiluminescence immunoassay on the Cobas e411 analyser. We estimated serum calcium and inorganic phosphate levels spectrophotometrically. Statistical analysis was performed utilizing SPSS version 26.0 by utilizing the Independent Sample t test and Pearson Correlation. In the hypothyroid patients, serum levels of Vitamin D3 had a mean value of 13.40 ± 3.95 ng/ml whereas the control group had a mean value of 31.15 ± 4.80 ng/ml. The probability value was <0.001. Moreover, calcium in hypothyroid patients was found to be significantly lower reaching 8.05 ± 0.62 mg/dl while phosphate was found to be significantly higher at 4.68 ± 0.75 mg/dl (p < 0.05). There was a strong positive correlation between the Vitamin D3 and the calcium with coefficient value of 0.712 and probability value was below 0.01. A significant negative correlation between Vitamin D3 and phosphate with a correlation coefficient of −0.585 and probability value < 0.01 was observed. Based on the findings of the study, it was concluded that older women with hypothyroidism significantly increase the risk of mineral deficiency and deficiency of Vitamin D3. Low thyroid level might disrupt the Vitamin D–calcium–phosphate axis and cause hypocalcemia and hyperphosphatemia. For the clinical management of thyroid diseases and prevention of secondary bone disorders, monitoring these biochemical markers should be done routinely.