The 34-year-old is sidelined for six weeks with a knee injury and will miss the summer showpiece in Poland and Ukraine.
Puyol underwent tests after Saturday's 4-0 win at home to Espanyol and the 34-year-old now needs arthroscopic surgery on his knee, which is set to keep him out for a period of around six weeks. A quicker comeback seems unlikely, especially with the defender now in his twilight years. For Spain at least, there is a real possibility that this is the end for one of the game's finest servants.
Puyol is a proud Catalan, but has graced the game for Spain since making his debut in 2000, putting his body on the line despite illness and injury to turn out for La Roja through thick and thin. In the good times and the bad, Spain has always been able to rely on Puyol, a veteran of three World Cups, two European Championships, one Olympic Games (in 2000) and a Confederations Cup (in 2009).
Always committed and fiercely competitive, the Barca defender has seen rivals come and go throughout the years, making one of the center back roles his own and acting as a captain of his country all but in name (that honor is held by Real Madrid's Iker Casillas).
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Having helped Spain claim its first silverware since 1964 with victory in Euro 2008, Puyol was inspirational once more as La Roja captured the World Cup two years later. The Catalan played a pivotal part in that triumph, rising majestically in the semifinal to crash home a thunderous header from Xavi's corner to seal a place in the South Africa showpiece for Vicente del Bosque's side.
Following that success, Puyol (then 32) claimed he would play on for Spain for at least two more years, but the subsequent season saw the defender sidelined for long periods by a troublesome knee problem which required surgery last summer and doubts emerged over his long-term fitness.
As usual, Puyol worked incredibly hard to return for Barca in the current campaign and Guardiola's team looked a much more solid side with him at the heart of the defense, although one or two uncharacteristic mistakes (against Real Madrid and Malaga recently) have crept into his game of late.
Now he will have to start from scratch once more. Puyol will want to return again - as he always does - but injuries finally appear to be taking their toll on his formidable frame. With 99 caps earned for La Roja, he may never reach that 100th appearance, and it seems even more unlikely that he'll hit that milestone this summer.
In the meantime, Spain must find a Plan B. With Puyol out, one option is to move Sergio Ramos across from right back to a central role. The Seville-born defender has been outstanding for Madrid this season as a center back and could play alongside Gerard Pique in the heart of the Spain back line.
Pique himself has had his troubles this term, but should start for La Roja this summer and if Ramos moves inside, Madrid's Alvaro Arbeloa will be favorite to start at right back.
Another option in that position is Athletic Bilbao's underrated Andoni Iraola, while his club colleague Javi Martinez is an additional alternative at center back, dropping deep from midfield with outstanding results for the Basques in 2011-12.
Whoever plays, Spain will remain strong, but replacing Puyol is impossible - both on and off the pitch. Del Bosque himself has described the loss as a "big setback". La Roja have lost a legend for the summer showpiece, and perhaps even forever. Football fans everywhere will hope that is not the case, that he can return, claim his 100th cap and go on to achieve more great things for both for his club and country. Because it wasn't supposed to end like this.
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