A new flattened species of Gracilariopsis from Mageshima Island, Japan is recognized based on morphological and molecular analyses of rbcL and cox1 DNA sequences. We name this new species Gp. mageshimensis sp. nov. The rbcL and cox1 analyses suggest that Gp. mageshimensis belongs to the Gracilariopsis clade, and that it is distinct from other Gracilariopsis species. Gracilariopsis mageshimensis is characterized by a flattened thallus, the lack of tubular nutritive cells, the lower gonimoblasts connect to the cystocarp floor cells via pit connections and small gonimoblasts filling the cystocarp cavity. The Gp. mageshimensis cystocarp is in accordance with the morphological concept of Gracilariopsis. Gracilariopsis mageshimensis is the second Gracilariopsis species with a flattened thallus to be described worldwide. Gracilariopsis mageshimensis is distinguished from Gp. silvana, the first flattened species by the rare production of lateral branches, a medulla consisting of fewer than five layers, and its deep-water habitat.