Mithramycin oxygenase OIV (MtmOIV) is a key enzyme of the biosynthetic pathway of the aureolic acid anticancer drug mithramycin (MTM). As the second last enzyme of the pathway, MtmOIV also has a major impact on the success of combinatorial biosynthetic studies towards new MTM analogues with novel functionality and possible reduced toxicity. MtmOIV is a 56 kD homo-dimeric FAD- and NADPH-dependent Baeyer-Villiger monooxygenase (BVMO), and likely the first BVMO investigated with its true natural substrate, the complex premithramycin B. We present the three-dimensional protein crystal structure of MtmOIV determined by X-ray crystallography using molecular replacement to a resolution of 2.9Å, and initial enzyme design studies aiming on broadening MtmOIV’s substrate specificity, which were based on the protein crystal structure. These studies could pave the way for the generation of a new generation of MTM anticancer drug analogues by combinatorial biosynthesis.