In the Irish parliament, Mr Flanagan said frontier controls would not be decided by London and Dublin alone if Britain voted to take itself out of the European Union.
“The outcome would be the result of a wider negotiation involving all of the EU and therefore no one can say with certainty that nothing will change with the border if the UK votes to leave,” he said.
His remarks will be seen as a rebuke to claims by Ms Villiers at the weekend that border arrangements between Northern Ireland and the Republic would not have to change in the event of a Brexit.
Ms Villiers said the land border would remain as “free-flowing” as it is today because of a special status for Irish citizens in the UK as well as the common travel area which pre-dates EU membership by both countries.
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The senior Tory minister also attacked suggestions that Brexit could threaten the peace process as “scaremongering of the most irresponsible and dangerous kind”.
But Mr Flanagan warned the “practically invisible border is a major symbol of normalisation and development in north-south relations”.