Wales fell to their first defeat to Japan after being outplayed in the heat of Tokyo.
Tom Prydie marked his return to international rugby after a three-year absence with a try but for the second week in a row, a second-string Wales were outscored two tries to one as Craig Wing and Michael Broadhurst touched down for Japan.
Full-back Ayumu Goromaru also kicked 13 points while Wales could only muster another Dan Biggar penalty.
It was an historic first win for Japan under Eddie Jones as they build towards the 2015 World Cup and hosting the tournament in 2019.
There was little sign of the shock to come as Wales dominated the opening 10 minutes but failed to make the most of their possession and territory.
Biggar was wide with his first attempt at goal in the third minute before Liam Williams lost possession as Wales edged their way towards the line, sparking a melee between both sets of forwards.
Harry Robinson threatened while lock Lou Reed almost created a chance when he charged down Japan fly-half Harumichi Tatekawa only for the ball to spin out of play.
Instead the hosts took the lead on 15 minutes when Goromaru kicked a penalty conceded by Rhys Gill in front of his own posts.
Wales equalised seven minutes later through Biggar that prompted a welcome water break amid the soaring temperatures.
Wales' forwards benefited from the interlude and after James King won a line-out, Lloyd Williams went close to twisting his way over.
Rhys Patchell replaced Owen Williams on 25 minutes after the centre failed to shrug off the effects of an earlier heavy challenge and almost finished a scything break by Robinson.
However, Japan finished the first half the stronger following bursts by captain Toshiaki Hirose and lock Hitoshi Ono before Goromaru kicked a second penalty from distance to lead 6-3 at half-time.
Wales regrouped at the break and were immediately on the front foot but Robinson was denied in the corner by Kenki Fukuoka.
The tourists grabbed the lead when Biggar and Patchell combined in midfield after Reed set up a forward drive and Biggar's floated pass found Prydie unmarked out wide for his second Test try.
Biggar pushed his conversion across the posts and Wales' advantage was limited to 8-6.
Japan struck back within minutes when Wing surged past Gill to score the hosts' second try.
Goromaru added the conversion from out wide and Japan were buoyant as they led 13-8.
Wales continued to squander chances and they were made to pay when Japan scored their third try.
Wing barged through new cap Josh Navidi before finding Broadhurst unmarked for his second try of the series against a tiring Wales.
Goromaru again converted from out wide and the large home crowd sensed an historic win.
Wales thought they had found a way back on 63 minutes when Patchell won the race to Biggar's kick.
However English referee Greg Garner ruled the young Welshman had failed to exert downward pressure before his second attempt that was beyond the dead-ball line.
It immediately brought to the fore the controversial decision not to have a television match official for the game though Garner was perfectly placed to make the decision.
Japan were buoyed and finished the far stronger of the teams before Goromaru added a fifth kick late on as the Brave Blossoms held out for a deserved victory.