Japan are now without a win in three matches following Tuesday's 2-1 defeat to Ukraine in Belgium.
Oleksandr Karavayev's strike midway through the second half secured the friendly win for Andriy Shevchenko's side in Liege.
Japan, who snatched a late equaliser in last Friday's 1-1 draw with Mali, found themselves behind again after 21 minutes when Naomichi Ueda deflected Yaroslav Rakitskiy's 25-yard strike beyond the wrong-footed Eiji Kawashima.
Vahid Halilhodzic's men responded before the break, with Tomoaki Makino rising unchallenged to head home Gaku Shibasaki's cross from the middle of the penalty area.
Ukraine were held 1-1 by Saudi Arabia in their last outing but found a way to regain their advantage on this occasion, as Karavayev drilled home from the edge of the box in the 69th minute following good work from Schalke's Yevhen Konoplyanka.
Japan piled on the pressure in the dying minutes, with substitute Yuya Kubo coming close and Shoya Nakajima seeing an injury-time free-kick saved by Andriy Pyatov, but Ukraine held firm to claim the victory.
