Aims: To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. Methods and Results: Ashbya gossypii spores were mutagenized by exposure to UV light and mutant ZP4 strain, producing riboflavin threefold the riboflavin that of the wild-type strain, was isolated by the first and second screenings. Proteomic analysis of ZP4 strain showed the expression patterns of eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain and those enzyme activities were investigated. When activated bleaching earth (ABE) containing 75 gl(-1) rapeseed oil was added in the culture of the ZP4 strain with oxygen-enriched air supplied, riboflavin concentration increased to 8.7 gl(-1) at 5 days of culture. Riboflavin production yield was 0.17 g g(-1) of consumed oil, which was eightfold higher than that of the wildtype strain. Conclusions: The results show that the mutant ZP4 strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source. Significance and Impact of Study: Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.