We aimed to determine whether vitamin D levels before prostate biopsy have diagnostic value for clinically significant prostate cancer. The study cohort included patients who underwent prostate biopsy. A total of 224 patients were enrolled in our study and serum vitamin D levels were measured from February 2016 to December 2019 in routine laboratory tests. To determine the relationship between vitamin D levels and aggressiveness of prostate cancer, we used logistic multivariate analysis. Based on the histopathological results of patients who underwent radical prostatectomy, the serum vitamin D level was significantly lower with the large tumor volume group. In the univariate analysis, the prostate cancer diagnosis rate was associated with low vitamin D levels. Low vitamin D level is negatively correlated with clinically significant prostate cancer (biopsy Gleason score of 7 or higher) in the univariate (Odds ratio [OR], 0.955; P < 0.001) and multivariate (OR, 0.944; P = 0.027) analyses. In conclusion, we found that the incidence of clinically significant prostate cancer might related to low vitamin D level in the Asian population. In the future, a larger population and prospective study are needed.