Objective To explore the role of serum 25-hydroxyvitamin D (25[OH]D) in the occurrence and recurrence of benign paroxysmal positional vertigo (BPPV) in patients with BPPV. Methods Patients with non-recurrent BPPV (n=207) and recurrent BPPV (n=42) were recruited from Department of Neurology of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine from Jan. 2013 to Dec. 2014, and patients with non-BPPV vertigo diseases in the same period were enrolled as controls (n=43). The recurrence of BPPV was determined within one year of follow-up. The differences of serum 25(OH)D levels between three groups were compared, and the factors influencing the levels of serum 25(OH)D of all subjects were analyzed. Results Compared with the control group, serum 25(OH)D levels in the non-recurrent BPPV and recurrent BPPV groups were significantly decreased (both P<0.01), while there was no significant difference between the non-recurrent BPPV and recurrent BPPV groups. The female proportion was increased in the recurrent BPPV group versus the other two groups, but the difference was not significant (P=0.073). The serum 25(OH)D level in female patients was significantly lower than that in male patients ([18.7±7.5] ng/mL vs[21.7±8.3] ng/mL, P=0.002). Multiple stepwise linear regression analysis showed that females(β=-2.6, P=0.005)and BPPV (β=-5.5, P<0.001) were the independent factors influencing the level of serum 25(OH)D. Conclusion There are gender differences in human serum 25(OH)D levels, and the level of 25(OH)D is decreased in patients with BPPV.