Massimiliano Allegri’s side, already on 84 points, knew a draw would be enough to wrap up the championship with six rounds remaining and took the lead in the first half through Moise Kean.
However, the home side fought back after the break as goals from Kevin Bonifazi and Sergio Floccari secured their first win against the Italian champions since February 1957.
The Turin club can still be named champions this weekend if second-placed Napoli fail to win away to rock-bottom Chievo on Sunday.
Allegri stood by his decision to pick a heavily rotated side, with an eye towards Tuesday's Champions League quarter-final second leg against Ajax in Turin, after the first leg ended 1-1.
It was Juventus’s second defeat of the league season and they remain 20 points clear of Napoli, while SPAL have risen to 13th place on 35 points, seven clear of the relegation zone.
Andrea Barzagli and Juan Cuadrado returned for Juve after injury lay-offs, while 17-year-old defender Paolo Gozzi and midfielder Grigoris Kastanos made their Serie A debuts for the club, as did substitute Stephy Mavdidi.
It was 19-year-old, on-form striker Kean who caught the eye again when he put Juve in front with an instinctive finish after 29 minutes.
However, SPAL were rewarded for an impressive start to the second half when Bonifazi rose to head in his team’s equaliser from a corner.
The hosts continued to play on the front foot and were rewarded with 15 minutes remaining when Floccari raced on to a loose ball in the area and swept a finish into the corner.
It was only SPAL’s second ever win against Juventus but marked their third successive home victory after they beat Roma and Lazio.
AC Milan strengthened their grip on fourth place in Serie A with a tense 1-0 win over Lazio at San Siro on Saturday (Apr 13) that ended with a scuffle breaking out between players from both sides .
Edin Dzeko scored his first home goal in almost a year to seal a 1-0 win for AS Roma over Udinese that moves them into fourth place in Serie A.
