Pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge with a survival rate of approximately 10%, largely due to the lack of diagnostic and therapeutic options. To identify novel therapeutic targets for PDAC, we previously developed a Drosophila melanogaster model, the ’ 4-hit ’ fly, which mimics the alterations in KRAS , TP53 , CDKN2A , and SMAD4 genes observed in PDAC. Through whole-body genetic screening using the 4-hit model, we identified riboflavin (RF) metabolism as a novel potential therapeutic target for PDAC. Additionally, transcript levels of four out of five genes that are involved in RF metabolism were elevated in human PDAC compared to non-tumor tissues. Roseoflavin (RoF), an antimetabolite of RF, resulted in a variety range of metabolic changes, decreased mitochondrial respiratory chain activity, and suppressed proliferation in cultured PDAC cells. Furthermore, the combination of RoF with the MEK inhibitor drug trametinib significantly suppressed the growth of human PDAC xenografts compared to either treatment alone. These findings underscore that dual targeting of RF metabolism and MEK represents a novel therapeutic strategy for PDAC.