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Background. Postoperative transient hypocalcemia (TH) is a common complication of total thyroidectomy. This retrospective study evaluated the clinical utility of preoperative vitamin D3 injection for the prevention of TH after total thyroidectomy. Methods. We included 2294 patients who underwent total thyroidectomy from January 2015 until October 2018 and retrospectively analyzed their data by complete chart review at our hospital. The patients were divided into two groups: vitamin D3 injection (VDI; n = 342) and vitamin D3 noninjection (VDN; n = 1952). TH was defined as serum calcium <8.2 mg/dL and signs or symptoms of hypocalcemia. Results. The mean preoperative serum 25-hydroxyvitamin D (25-OHD) levels of the VDI group were significantly lower than those of the VDN group (16.5 ± 6.9 ng/mL vs 19.4 ± 8.7 ng/mL, p < 0.001). Multivariate analysis indicated that the significant risk factors of TH include vitamin D noninjection (hazard ratio (HR): 1.717, 95% confidence interval (CI): 1.282–2.300, p < 0.001), male gender (HR: 1.427, 95% CI: 1.117–1.822, p = 0.004), and capsular extension (HR: 1.273, 95% CI: 1.011–1.603, p = 0.040). Conclusions. Preoperative vitamin D3 injection significantly contributed to the prevention of TH after total thyroidectomy. Further prospective or multicenter studies must be conducted to determine the effect of vitamin D3 injection.