Outside the Tokyo courthouse on Friday, the plaintiffs of the lawsuit and their legal team held up signs that read "unjust verdict", local media reported.
Shino Kawachi, one of the plaintiffs, told local media that the ruling was "difficult to comprehend".
"What is justice? Was the court even watching us? Were they considering the next generation?" she told local media.
Her partner, Hiromi Hatogai, said she was "extremely outraged" and wondered if the judiciary was "on our side". But, she added, they would "keep fighting".
Amnesty International described the ruling as a "damaging step backwards on same-sex marriage".
"The Japanese government needs to be proactive in moving towards the legalisation of same-sex marriage so that couples can fully enjoy the same marriage rights as their heterosexual counterparts," the rights group's East Asia researcher Boram Jang said in a statement.
The verdict on Friday is the last among six high court rulings on same-sex marriage lawsuits filed between 2019 and 2021, in courts across the country from Sapporo to Osaka to Fukuoka.
Among them, five found the ban unconstitutional - though they rejected the plaintiffs' compensation claims.
Friday's ruling by the Tokyo high court marks an exception to this series of verdicts, which had been adding weight to the push for same-sex marriage to be legalised in Japan.
The cases will next be taken to the Supreme Court.