"If we allow Catalonia -- though it's not our business -- to separate itself from Spain, others will do the same. And I wouldn't like to have that," Juncker said in a speech to students in his native Luxembourg.
"I wouldn't like to have a European Union which consists of 98 states in 15 years' time. It's already relatively difficult at 28, no easier at 27 (after Britain leaves), but at 98, that seems impossible."
Juncker said he had urged Spanish Prime Minister Mariano Rajoy to "for a long time take the initiative so that the situation in Catalonia did not become what it has now."
Brussels has insisted the dispute over Catalonia's independence referendum is an internal matter for key EU member Spain, backing Madrid's position that it was illegal, but calling for dialogue.
Juncker added that the EU "cannot involve itself into mediation" as Catalan leader Carles Puigdemont has called for, and Madrid has rejected.
Madrid has given Puigdemont until next Thursday to abandon his push for secession, failing which it may trigger unprecedented constitutional steps that could see Madrid take direct control of the semi-autonomous region.
Puigdemont's separatist allies pressed him today to defy Madrid and declare independence.
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