The EU Commission chief said Scotland would have to go through the “same procedures” as any other country looking to join the club in a shattering blow to the SNP chief’s independence dream.
Ms Sturgeon has built her drive for separation firmly on the argument that Edinburgh would be able to keep the newly independent nation inside the EU and its lucrative Single Market.
But Brussels has always stressed this would not be possible, stating that Scotland would have to leave the bloc with the rest of the UK and then re-apply for membership as a new country.
The last country to join the bloc, Croatia, had to wait 10 years between its application and its actual accession, although Scottish politicians would argue they have convergence with EU standards from the get-go due to the UK’s membership.
Eurocrats are particularly determined not to make any exceptions to their membership rules due to the sensitivities of Spain, where the region of Catalonia is set to hold a referendum on independence that Madrid deems illegal.
Most countries in the Europe face secessionist movements of varying sizes and influence, and any suggestion the EU would smooth the path to membership could spark a catastrophic wave of break-offs.
Asked about the Catalan situation today, Mr Juncker said: “If there were to be a yes vote in favour of Catalan independence then we will respect that opinion.
“But Catalonia will not be able to become an EU member state on the day after such a vote.
“It will have to follow the same accession procedures as those member states did who joined in 2004.”
Pressed on whether the same principle applies to Scotland, he categorically replied: “Yes, and if the north of Luxembourg were to secede from the south the rules would apply to that too.”
And despite claims from some SNP figures that Brussels is sympathetic to their independence cause, the EU boss made clear that he is not a fan of regions splitting off and going it alone.
He said: “Europe has a wealth of different regional traditions. This is part of the richness of Europe, but I do not want regional traditions to lead to separatism and fragmentation of Europe.”
The issue of Scottish independence has died down in recent months, following a flurry of activity in the immediate aftermath of the Brexit vote when Ms Sturgeon tried to capitalise on the fact that 62 per cent of Scots backed Remain.
The SNP leader made a bold call for a second referendum – just three years after the “once in a generation” first – but was forced into a humiliating climbdown following a lukewarm public reception.
She even travelled to Brussels in an attempt to persuade EU leaders to back her cause, but was brutally snubbed by the EU Council chief Donald Tusk who refused to meet her.
The latest opinion polls show the independence movement is lagging 13-14 per cent behind, with support for the union having actually increased on its levels before the Brexit referendum.
And on top of that Ms Sturgeon’s credibility took a battering as the SNP suffered a bruising result in June’s General Election, losing 21 seats to pro-Union parties like the Conservatives and Labour.