Background and objective: Premature ejaculation is one of the most common and complex sexual diseases among men. Although there are four defined subtypes of premature ejaculation, the most common subtype is acquired premature ejaculation. This study investigated the association between serum vitamin D levels and acquired premature ejaculation. Material and methods: This retrospective cross-sectional study included 94 patients with a complaint of acquired premature ejaculation (the study group) and 92 patients without a complaint of premature ejaculation (the control group) between June 2018 and March 2020. Patients’ demographic characteristics, laboratory results, intravaginal ejaculatory latency time, Turkish validated premature ejaculation diagnostic tool (PEDT), International Index of Erectile Function-5 (IIEF-5) and Beck Depression scores were statistically compared between groups. Results: The patients’ mean age was 49.10 \pm 14.85 years. There were differences between the two groups regarding serum vitamin D levels, dehydroepiandrosterone sulfate, total testosterone, and follicle-stimulating hormone levels. Upon comparing the questionnaire scores, the PEDT, intravaginal ejaculatory latency time, and Beck depression scores were clinically significant between groups. The cut-off value for serum vitamin D levels was 8.37 ng/mL, with a sensitivity of 93% and specificity of 92%. Conclusion: We revealed that serum vitamin D and follicle-stimulating hormone levels were independent risk factors according to the multivariate analysis. Measurement of serum vitamin D levels in patients with acquired premature ejaculation may be useful for treatment.