We found that the marine yeast strain W14-3 isolated from seawater of China Eastern Sea could produce riboflavin. It is interesting to observe that the marine yeast strain produced a large amount of riboflavin in the medium containing xylose, sucrose, galactose and maltose under the conditions of vigorous shaking. The yeast strain was found to belong to Candida membranifaciens subsp. flavinogenie based on the results of routine and molecular identification. The protein sequences deduced from the partial genes encoding GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase in the yeast exhibited high identity with those of the corresponding enzymes for riboflavin biosynthesis in other yeasts. Fe(3+) available in the medium repressed riboflavin production and expression of the genes responsible for riboflavin biosynthesis in the yeast. The results have evidenced that a riboflavin synthesis pathway indeed existed in the yeast. This is the first study to report that C. membranifaciens subsp. flavinogenie W14-3 from the marine environment could produce riboflavin.