Like many other gobies, male Isaza (Gymnogobius isaza) which are endemic to Lake Biwa, Japan, exclusively care for broods in nests. This goby may have an optimal range of brood size (i.e., an average clutch size of about 2000-3000 eggs) within which they may produce larger numbers of hatching young because much larger broods may be destroyed by fungal infection before hatching. This optimal brood size hypothesis (Takahashi et al. in J Ethol 22:153-159, 2004) predicts that (1) after spawning, both males and females will refuse additional spawning by other gravid females (second females) to keep brood sizes within optimal ranges, (2) larger fish will repel second females more successfully than will smaller fish, and thus, (3) both sexes prefer larger mates. To examine these predictions, we first observed Isaza's aggressive behaviors in aquaria and investigated whether fish attacked and repelled second females that were introduced after spawning, and, if so, what were the sizes of fish that did so.Large fish, regardless of sex, aggressively prevented second females from entering the nest, but second females larger than the pairs displaced the pair females forcibly and spawned eggs into their clutches. Mate choice experiments showed that males preferred large females. Although females' choice of large mates was not confirmed, many results may largely coincide with the predictions of the optimal brood size hypothesis. Thus, Isaza males' choice of large mates will be advantageous for defending against brood parasitism by conspecific females and for achieving optimal clutch size.