In freezing conditions in Pamplona, Osasuna striker Dejan Lekic scored twice inside the first 22 minutes and Barca, who have struggled on the road all season, waited too long to react.
Spanish World Cup winners Xavi, Andres Iniesta and Cesc Fabregas started on the bench, and although Alexis Sanchez pulled one back for the visitors in the 52nd minute, Raul Garcia quickly restored Osasuna's advantage.
Barca's youth team winger Cristian Tello grabbed a second in the 73rd but a late charge just failed to produce the equaliser.
The world and European champions' second defeat of the season left them on 48 points.
"The league is much tougher now, and I suppose tomorrow we will see the gap grow even larger, " Barca coach Pep Guardiola told reporters.
"If we could play well on this pitch in the second half we should have done so in the first as well. We found it hard to get into the game."
Osasuna are seventh also with 31 points, one ahead of King's Cup finalists Athletic Bilbao, who suffered a 2-1 defeat at Real Betis in the late game.
Osasuna's bumpy pitch and a swirling wind saw both sets of players struggling to keep their feet at the Reyno de Navarra stadium, but the hosts adapted quicker than Barcelona.
A poor clearance from Valdes, one of a string of errors among Barca's players, led to Osasuna's third, when Raul Garcia squeezed a shot in off the post in the 55th.
