On a dramatic, rain-lashed night at the Camp Nou, the Portuguese forward's 73rd-minute strike from Mesut Ozil's superb assist secured the win his coach and compatriot Jose Mourinho had sought in vain in eight previous trips to Barca's imposing arena during his otherwise glittering career.
It was Real's first success in eight La Liga 'Clasicos' - four of them before Mourinho arrived from Inter Milan - and gives them a timely boost for Wednesday's Champions League semi-final second leg at home to Bayern Munich, when they will seek to overturn a 2-1 deficit.
"We have struck a very significant blow but it's not yet decisive," Real midfielder Xabi Alonso said in an interview with Spanish television.
"Of course it's an important step towards the title but now we have to see it home," added the Spain international.
"Cristiano's goal, coming straight after they levelled, broke their stride and that was fundamental.
"We controlled the match perfectly. It was a great performance, a fantastic result and I am very happy. Now we have another key match on Wednesday."
Barca, by contrast, need to regroup after suffering back-to-back defeats for the first time in almost three years following Saturday's reverse and a 1-0 loss at Chelsea in their Champions League semi-final first leg on Wednesday.
Pep Guardiola, whose only defeat in 14 previous 'Clasicos' as Barca coach was in last year's King's Cup final, may also have his tactics questioned after he left Alexis Sanchez, Cesc Fabregas and Pedro out of his starting line-up.
Sanchez came off the bench to snatch a 70th-minute equaliser after a rare Sami Khedira goal had given Real an early lead, while a toothless Barca could have done with Fabregas's creativity and eye for goal much earlier than the 80th minute.
Guardiola opened his news conference by conceding his bid to lead Barca to a fourth straight La Liga crown had failed.
"I congratulate Real Madrid on their victory and on the league title," the former Barca and Spain midfielder said.
"Right now the players are tired and sad and I can't say if there will be a psychological effect for the Chelsea game," he added.
"We will try to find a way to attack Chelsea but now we have to rest and recover and try to forget tonight's game as quickly as possible."
As the two teams walked out for a match watched by hundreds of millions around the world, close to 100,000 home fans transformed the stadium into a giant mosaic in the blue and red colours of the club and the yellow and red of Catalonia.
"Som i serem" (We are and we will be) was picked out in yellow on one side of the arena as the home support bellowed the club anthem "El Cant del Barca".
Guardiola deployed an attacking formation with only three defenders and featuring the surprise inclusion of pacy 20-year-old B-team winger Cristian Tello.
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