Madrid is seeking to stop the Catalan government spending public money on its planned independence referendum.
Juncker was asked if the European Commission would recognise and accept a "Yes" result in the 1 October referendum in Catalonia.
Despite warnings from the federal government in Madrid that the referendum contravenes the Spanish constitution, the referendum campaign was launched Thursday by pro-independence groups in Tarragona.
This new system will allow the Spanish state to "replace the region for the majority of essential spending", he added.
President of the Catalan Government Carles Puigdemont (R) signs a decree calling independence referendum past Catalan regional vice-president and chief of Economy and Finance and leader of the Esquerra Republicana de Catalunya (ERC) leftist Republican Party Oriol Junqueras at the Catalan Parliament in Barcelona, on September 6, 2017.
He said the arrangement implied "political control that is not related to the objectives of budget stability or to the purposes of state legislation in this matter". The central government had ordered them in July to scrutinize that public funds were not being used on the illegal vote.
The appeal comes at the end of a week in which the state prosecutor summoned mayors for questioning, and Catalonia rebelled against Madrid's fiscal rules.
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The decision follows a series of moves to block a Catalan referendum on self-rule, planned for October 1, which Prime Minister Mariano Rajoy's government has declared illegal.
"We have reached this moment stronger than what many had thought and wanted, as we are proving by responding firmly to each threat", he said.
"We call for. open and unconditional dialogue".
"But Catalonia will not become an European Union member state the day after that vote".
The signatories also accused the Spanish administration, lead by Mr Rajoy of having gone "on the offensive with unprecedented repression".
Madrid has the constitutional power to take over a regional government or send in the police to force Catalonia to drop the vote, but either step would rock Spain's decentralised model of government where power is devolved to 17 self-governed regions.
Supporters of independence are celebrating already despite surveys indicating less than 50 percent of Catalonia's population want full self-rule.