Objective: To determine associations between vitamin B status, homocysteine (tHcy), semen parameters, and sperm DNA damage. Design: Observational study. Setting: A tertiary referral fertility clinic. Patient(s): Two hundred fifty-one men of couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment, with subgroups of fertile (n = 70) and subfertile men (n = 63) defined according to semen concentration and proven fertility. Intervention(s): None. Main Outcome Measure(s): The DNA fragmentation index (DFI) as marker of sperm DNA damage determined using the sperm chromatin structure assay (SCSA), and semen parameters assessed according to World Health Organization criteria; tHcy, folate, cobalamin, and pyridoxine concentrations determined in seminal plasma and blood. Result(s): In the total group of fertile and subfertile men, all biomarkers in blood were statistically significantly correlated with those in seminal plasma. No correlation was found between the biomarkers in blood and the semen parameters. In seminal plasma, both tHcy and cobalamin positively correlated with sperm count. Folate, cobalamin, and pyridoxine were inversely correlated with ejaculate volume. In fertile men, seminal plasma folate showed an inverse correlation with the DNA fragmentation index. Conclusion(s): Low concentrations of folate in seminal plasma may be detrimental for sperm DNA stability.