Background. Total thyroidectomy (TT) is recommended in the treatment of malignant and benignant thyroid diseases, and, to date, transient hypocalcemia is the most frequent complication after the procedure. We prospectively evaluated the role of vitamin D deficiency as a predictor of postoperative hypocalcemia. Methods. This is a prospective cohort study which was conducted between January 2016 and April 2019. A total of 177 consecutive patients (141 (79.7%) women and 36 (20.3%) men) who underwent TT were included in the current study. Hypocalcemia occurred when serum calcium levels were below 8.0 mg/dL or 1.10 mmol/L. Patients were divided into two groups (Group 1, normocalcemic; Group 2, hypocalcemic) and were assessed taking into consideration preoperative serum 25-hydroxy vitamin D (25-OHD) levels, preoperative serum calcium levels, thyroid hormone levels, sex, body mass index (BMI), and smoking habits. Vitamin D deficiency was defined as 25-OHD levels <25 ng/mL. Results. The incidence of postoperative asymptomatic and symptomatic hypocalcemia in the two groups was 19.8% and 15.8%, respectively. Preoperative 25-OHD level was significantly different between Group 1 and Group 2 (31.5 ± 15.0 ng/mL vs 18.7 ± 9.8 ng/mL,p=0.017). Logistic regression analysis revealed that preoperative vitamin D deficiency was a significant predictive factor of postoperative hypocalcemia (p=0.012), and, specifically, the risk of hypocalcemia increased 15-fold in patients with a preoperative vitamin D level <25 ng/mL (odds ratio [OR], 14.8). Conclusions. Postoperative hypocalcemia is significantly associated with low preoperative levels of serum 25-OHD. Our studies demonstrate that vitamin D deficiency (<25 ng/mL) is an independent predictive factor of postoperative hypocalcemia.