The Blaugrana suffered their second league defeat in 47 matches when they failed to complete the comeback after a first half flurry from the home side.
A first half brace by Dejan Lekic gave Osasuna a magical start. The second half saw a flurry of goals as Alexis Sanchez reduced the deficit, only for Raul Garcia to add to it once more. Cristian Tello made it 3-2, but Barcelona could not complete the comeback.
The Catalan side began the match with a revolutionized starting 11 as Sergi Roberto, Pedro, and Thiago Alcantara started in place of Xavi, Andres Iniesta, and Cesc Fabregas.
The changes hurt the La Liga giant early on as Osasuna got off to a dream start. In the fourth minute, Garcia received the ball on the edge of the 18-yard box and flicked on to find a sprinting Lekic who buried his strike past Victor Valdes to open scoring.
Pep Guardiola's men attempted to fight back but struggled to establish their quick, one-touch game with the frozen pitch.
The home side would not sit back and attempt to protect the lead as so many sides have failed to do. After being picked out by Garcia on the right flank, Alvaro Cejudo looped in a cross which found a surging Lekic who tapped past Valdes to shockingly double his side's lead and earn a brace.
The first half ended with the Blaugrana being able to establish dominance in possession but not being able to create any goal-mouth opportunities with it.
When the second half began Guardiola made a few changes, but not what was expected. Instead of bringing Iniesta, Xavi, or Fabregas into the game, he introduced Isaac Cuenca and Tello.
Although the changes may have confused a few fans, they worked. Cuenca centered the ball from the right and found an unmarked Sanchez who slid the ball past Fernandez to bring the away side back into the game.
Los Rojillos did not tremble and instead marched towards Barcelona's goal, once again to be rewarded. After a pass from Valdes was intercepted, Nino was picked out on the left. The experienced forward sent in a cross and Garcia managed to tip past Valdes, punishing the keeper for his error.
The match continued to see-saw back and forth as La Catalunya reduced the lead once more. After being picked out on the right, Tello made short work of his marker and blasted past Fernandez from short range to once again augment his side's aspirations.
Barcelona looked to have equalized when Sanchez found the back of goal from a corner kick, but was adjudged to be offside by the linesman. The away side pressured its opponent, but could not complete the comeback.
With the loss, Barcelona can see any aspirations of winning La Liga slip away as it could find itself 10 points off of first with a Real Madrid victory on Sunday. Osasuna has pulled off a fantastic upset victory and now finds itself in seventh place.