We isolated protoplasts from male and female gametophytes of a strictly dioecious strain of the coenocytic marine green alga Bryopsis plumosa. The protoplasts successfully developed into macrothalli. These in turn produced swimming cells, which appeared similar to biflagellated gametes even when the mixed protoplasts were comprised of protoplasm from male and female gametophytes. We found that swimming cell sizes depended on the male/female protoplasm ratio; macrothalli successfully produced swimming cells with male/female protoplasm ratios of 10:0; 9:1; 7:3; 5:5; 1:9; and 0:10. In male/female protoplasm ratios ranging from equal to strongly female biased (5:5; 3:7; 1:9), swimming cells exhibited normal behaviors of gametes and resultant zygotes, displaying positive and negative phototaxis, respectively. Negatively phototactic swimming cells were quadriflagellated and had two nuclei, apparently as a result of fusion, but never developed into microthalli. Thus, these swimming cells might lack functionality essential for normal gametes. Our findings suggested that natural monoecy observed in this genus did not originate from hybridization of protoplasm between the sexes.