Hosts Britain, making their first appearance at the Games for 52 years, were denied a win in front of a 72,000 crowd at Old Trafford when Senegal scored a late equaliser in a 1-1 draw.
Uruguay, back in the Games after an 84-year absence, came from behind to beat a lively United Arab Emirates side 2-1 in the earlier game at Old Trafford.
The Spaniards finished the Group D match with 10 men after midfielder Inigo Martinez was sent off just before half-time having found no answer to Yuki Otsu's 34th minute goal.
Asked if this was Japan's best ever win coach Takashi Sekizuka said: "We looked forward to the match very much and showing how much we have to offer was the main point.
"Now we have to prepare for winning the games against Morocco and Honduras but I am very pleased with the result today."
Despite Spain's hopes of winning the title, Brazil are favourites to take their first Olympic football gold.
Neymar, who scored from a header, told reporters: "We need to improve, but it is always better to improve for the next match after a win."
Substitute Nicolas Lodeiro scored Uruguay's winner while captain Luis Suarez was booed every time he touched the ball by a Manchester crowd unforgiving of the Liverpool player's racial spat with United's Patrice Evra last season that earned him a lengthy ban.
Suarez said: "This is just one more match. I'm used to playing in stadiums where I'm booed. It doesn't worry me in the least. They boo me at almost every stadium in England, it's normal. I'd rather highlight the support from the Uruguayan fans."
In Thursday's other matches, Morocco and Honduras
