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Abstract Background Benign paroxysmal positional vertigo is the most common neurootologic disorder, characterized by brief attacks of vertigo aggravated by certain head positions. During the last two decades, several studies were performed in different countries trying to find a relationship between benign paroxysmal positional vertigo and vitamin D3 (25 hydroxyvitamin D) deficiency. Objectives This study aimed to find the relationship between vitamin D3 deficiency and total serum calcium with benign paroxysmal positional vertigo. Patients and methods A case–control study conducted from January 2021 to December 2021, consisted of 62 participants; 30 patients with posterior canal benign paroxysmal positional vertigo and 32 healthy persons considered as a control group. The age and sex of both groups were matched, and diagnosis was made by history and examination (Dix-Hallpike’s maneuver). A blood sample was taken to evaluate serum vitamin D level and total serum calcium. The results were compared between both groups. Results The mean levels of vitamin D3 in patients and control groups were 18.57 ± 9.88 ng/ml and 64.12 ± 24.64 ng/ml, respectively, with a statistically significant difference at p ≤ 0.0001. Moreover, regarding vitamin D3 deficiency below 20 ng/ml between patients and control groups, there was a statistically significant difference p = 0.001. Furthermore, there was no significant difference regarding total serum calcium between the two groups, the mean of both groups was 8.57 ± 0.68 mg/dl and 8.93 ± 0.92 mg/dl for patients and control, respectively, at p = 0.084. Conclusion There might be an association between benign paroxysmal positional vertigo and vitamin D3 deficiency. Moreover, there was no significant difference concerning total serum calcium levels between the patient and control groups.