We examined the association between 24-h urinary excretion of water-soluble vitamin levels and their intakes in free-living Japanese university students. The design used was cross-sectional study. A total of 216 healthy, free-living male and female Japanese university students aged 18-27 years voluntarily participated in this study, of which 156 students were eligible for this assessment. All foods consumed for 4 consecutive days were recorded accurately by a weighed food record method. A 24-h urine sample was collected on the fourth day, and the urinary levels of water-soluble vitamins were measured. Each urinary water-soluble vitamin level, except for vitamin B(12), was correlated positively with its mean intake in the recent 2-4 days (vitamin B(1): r=0.42, P<0.001; vitamin B(2): r=0.43, P<0.001; vitamin B(6): r=0.40, P<0.001; vitamin B(12): r=0.06, P=0.493; niacin: r=0.35, P<0.001; niacin equivalents: r=0.33, P<0.001; pantothenic acid: r=0.47, P<0.001; folate: r=0.27, P=0.001; vitamin C: r=0.44, P<0.001). Mean estimated water-soluble vitamin intakes calculated from urinary levels and recovery rates showed 91-101% of their 3-day mean intakes, except for vitamin B(12) (61%). These results showed that urinary water-soluble vitamin levels, except for vitamin B(12), reflect their recent intakes in free-living Japanese university students, and could be used as a potential biomarker to estimate mean vitamin intake.