Spanish Prime Minister Mariano Rajoy has warned Scotland has no right to negotiate with the EU.
The ultra-unionist Conservative declared: "If the the United Kingdom leaves, then Scotland leaves too."
Read more: Nicola Sturgeon holds Brussels talks to set out Scotland's EU position
His intervention came as Nicola Sturgeon prepares for talks with European Commission President Jean-Claude Juncker in Brussels in an historic diplomatic breakthrough.
The First Minister has failed to secure a meeting with Donald Tusk, the Polish president of the European Council, while other states, including Germany, signaled they would not commit to talks.
Mr Rajoy added: "Scotland has no powers to negotiate with the European Union."
Read more: Nicola Sturgeon holds Brussels talks to set out Scotland's EU position
Brussels watchers have long expected Spain to "huff and puff" - as once source said - over Scottish membership of the European Union.
The president of Catalonia - which has been barred from holding its own independence referendum by Mr Rajoy and his government - on Wednesday declared that the EU's attitude had changed.
Carles Puigdemont said: "The EU will radically change its idea about the independence of Scotland and Catalonia" because of Brexit.
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