The president of the executive European Commission made the remarks during a broadcast on Youtube on Thursday.
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 be an EU member state on the day after such a vote.”
Catalonia’s parliament has laid the ground for a referendum on independence from Madrid on October 1, although Spain’s Constitutional Court has suspended the vote.
Judges are now considering whether the legislation contravenes Spain’s constitution.
Echoing a stance made by his predecessor, Jose Manuel Barroso, Mr Juncker said any newly independent state must follow the same EU membership procedures as all aspirants, citing Scotland and, in jest, his home country, Luxembourg.
Mr Juncker said: “If northern Luxembourg were to cede from the south, the same rules would apply.”
Catalonia is one of Spain’s 17 autonomous regions. Its capital is the dynamic, touristic and cultural Mediterranean port city of Barcelona.
The region has 7.5 million inhabitants and is one of Spain’s main economic powerhouses, generating a fifth of the country’s €1.1trillion economy.
It has its own language, which was suppressed during the 1939-1975 dictatorship of General Francisco Franco, and cultural traditions. It is also home to one of the world’s greatest soccer teams, FC Barcelona.
While many Catalans have long stressed the region’s differences from the rest of Spain, the current push for independence began in earnest 2010 when Spain’s Constitutional Court struck down key parts of a groundbreaking charter that would have granted Catalonia greater autonomy and recognised it as a nation within Spain.
The court’s rejection was felt bitterly in the region and has since driven hundreds of thousands of residents out onto the streets every September 11, a Catalan holiday, to demand independence.
The Catalan independence row has also led Spain’s top prosecutor to investigate more than 700 Catalan mayors for cooperating with a planned referendum on the region’s independence after the nation’s constitutional court ordered the vote put on hold.
Catalonia’s regional police force is under orders to arrest the mayors if they refuse to appear for questioning, State Prosecutor Jose Manuel Maza’s office said.
The pro-independence coalition ruling Catalonia has vowed to hold the referendum, defying a prohibition by Spain’s Constitutional Court.
It has asked the 947 mayors in the northeastern region to provide voting facilities.