Phenytoin, an antiepileptic drug, induces neurotoxicity and abnormal embryonic development and reduces spontaneous locomotor activity in fish. However, its effects on other endpoints remain unclear. Therefore, we investigated the effects of phenytoin on the swimming behavior and reproductive ability of Japanese medaka. Abnormalities in swimming behavior, such as imbalance, rotation, rollover, and vertical swimming, were observed. However, when phenytoin exposure was discontinued, the behavioral abnormality rates decreased. Phenytoin exposure also significantly reduced reproductive ability. By investigating reproduction-related gene expression of gnrh1, gnrh2, fshb, and lhb remained unchanged in males and females. In contrast, kiss1 expression was significantly suppressed due to phenytoin exposure in males and females. kiss2 expression was also significantly suppressed in females but not in males. We filmed videos to examine phenytoin exposure effects on sexual behavior. Females showed no interest in the male's courtship. As the kisspeptin 1 system controls sexual behavior in Japanese medaka, phenytoin exposure may have decreased kiss1 expression, which decreased female reproductive motivation; hence, they did not spawn eggs. This is the first study to show that phenytoin exposure induces behavioral abnormalities, and suppresses kiss1 expression and reproductive performance in Japanese medaka.