Injuries to Fernando Torres and Gerard Pique capped a miserable night for Spain as they slipped to a rare defeat against Italy in Bari.
Riccardo Montolivo netted after 11 minutes to cap a good start by Italy, before Chelsea striker Torres was withdrawn, apparently suffering from concussion.
Xabi Alonso levelled from the spot eight minutes before the break, but the night took a turn for the worse for the Spanish as Pique was also withdrawn and Liverpool misfit Alberto Aquilani grabbed a winner six minutes from time.
Italy almost got off to a perfect start when Domenico Criscito struck the post from 25 yards after four minutes.
Andrea Ranocchia fired over after 11 minutes, but their pressure paid off less than a minute later as Criscito found Montolivo, who beat Iker Casillas from the edge of the area.
Things got worse for Spain in the 15th minute when Torres was withdrawn, replaced by Fernando Llorente. It was reported later Torres had suffered mild concussion and was taken to hospital as a precaution.
David Silva brought a save out of Italy keeper Gianluigi Buffon, while at the other end Casillas kept out Giuseppe Rossi's effort from point-blank range.
Andoni Iraola and Santi Cazorla fired wide for Spain, before Giorgio Chiellini brought down Llorente in the box.
Alonso converted from the spot, but it continued to be Italy who looked the more threatening as Antonio Cassano was first denied by Casillas and then twice headed over.
There was further injury woe for Spain before the break when Barcelona defender Pique, who appeared to have picked up a thigh injury, was replaced by Sergio Busquets.
Spain switched goalkeepers at half-time, with Victor Valdes replacing Casillas, but it did little for Cassano as the Barcelona man produced to a save to keep out his 12-yard effort.
Buffon fumbled Llorente's shot on the hour but it did not prove costly.
David Silva forced Buffon to parry 12 minutes from time, but it was Italy who got the winner when substitute Aquilani picked up Giampaolo Pazzini's pass six minutes from time and buried the ball past Valdes.
Mario Balotelli had a chance to finish things off late on but Valdes saved with his feet.