We investigated serum folic acid (FA) levels in patients with erectile dysfunction (ED) and/or premature ejaculation (PE). Fasting serum samples were obtained from 42 patients with ED, 36 with PE, 25 ED patients with PE, and 30 healthy men; the mean intravaginal ejaculation latency time (IELT) was measured during a 4 weeks baseline period. Levels of sex hormones (follicle-stimulating hormone, luteinizing hormone, total testosterone), homocysteine (Hcys), and FA were measured using chemiluminescent immunoassays. The sexual functions of PE patients and normal control men were evaluated using the Chinese Index of Premature Ejaculation (CIPE). The abridged International Index of Erectile Function-5 (IIEF-5) questionnaire was used to gauge erectile quality for ED patients and for normal controls. Serum FA concentrations were lower in ED (7.61 ± 3.97 ng ml⁻¹), PE (9.37 ± 3.40 ng ml⁻¹), and ED/PE (8.84 ± 4.28 ng ml⁻¹) patients than in healthy men (12.23 ± 5.76 ng ml -1 , P < 0.05). No significant differences in sex hormone levels were found between patients with sexual dysfunction and healthy controls (P > 0.05). There were positive correlations between serum FA concentrations and CIPE scores (r = 0.530, P < 0.01), IIEF-5 scores (r = 0.589, P < 0.01), and IELT (r = 0.445, P < 0.01); negative correlations with Hcys concentrations (r = -0.487, P < 0.01) were found in all participants. These findings showed a strong relationship between serum FA levels and sexual dysfunction, possibly due to an effect of FA on the metabolism of nitric oxide, Hcys, and 5-hydroxytryptamine.