Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG.