Macrocyclic formation in natural products is typically understood to proceed through direct macrocyclization reactions. Here we demonstrate that complex macrocycles can instead be constructed through stepwise ring expansion of a pre-formed smaller scaffold. Genetic and biochemical analyses of luminamicin (1) biosynthesis revealed that a 10-membered ring is progressively transformed into a 14-membered macrocycle through two mechanistically distinct ring expansion steps. This process involves a cofactor-independent intramolecular acyl transfer and a radical-mediated oxidative rearrangement, which together reorganize a pre-assembled intermediate into the final macrocyclic framework. This stepwise remodeling strategy contrasts with canonical macrocyclization logic and provides a solution to the entropic and conformational constraints associated with the formation of large rings. More broadly, these findings establish stepwise ring expansion as a previously unrecognized biosynthetic strategy for constructing complex macrocycles and suggest new strategies for their chemical synthesis.