A filamentous fungus Ashbya gossypii overproduces riboflavin. However, what is a trigger to produce riboflavin in A. gossypii has not yet been elucidated. In the present study, we reveal the mechanism, by investigating riboflavin production in A. gossypii with respect to endoplasmic reticulum (ER) stresses. Addition of a reducing agent, dithiothreitol (DTT), to the culture medium at 10 mM resulted in more yellowish mycelia compared to the that of wild type strain, enhanced specific riboflavin production by approximately 1.7-fold, and an increase in the expression of several rib genes, AgRIB1, AgRIB2, and AgRIB7. Additionally, the production of reactive oxygen species (ROS) and expression of antioxidant genes, AgGLR1 and AgSOD1, were also induced. The ER stressor, tunicamycin, which inhibits the addition of N-glycan to glycoproteins in the ER, also enhanced the riboflavin production by 1.8 fold with the production of ROS and the increase of the expression of AgRIB3 and AgRIB4 genes. These results indicate that ER stress enhances riboflavin production in A. gossypii through the production of ROS; however, the mechanism of ER stress-induced riboflavin production has not yet been revealed. This study reveals why A. gossypii naturally overproduces riboflavin. This study shows the different mechanisms of the riboflavin over-production by ER stressors, DTT and tunicamycin. Additionally, cycloheximide, which inhibits protein synthesis and induces apoptosis, enhanced the riboflavin production. These results suggest that apoptosis induced by ER stresses may partially enhance the riboflavin production in A. gossypii. DTT and tunicamycin can be utilized to reveal why the riboflavin overproduction is conducted in A. gossypii, leading to the generation of riboflavin-hyperproducing A. gossypii mutants for efficient industrial riboflavin production.