Nicola Sturgeon has asked the EU to “keep the light on” for Scotland and that it will be “back soon”.
The first minister tweeted the message shortly after the Brexit transition came to an end at 11pm on New Year’s Eve.
Scotland’s trading and travel relationship with EU countries will now be governed by the deal agreed by London and Brussels on Christmas Eve.
Ms Sturgeon’s Scottish National Party (SNP) wants to see an independent Scotland join the EU.
In the 2016 referendum, 62% of those who voted in Scotland backed Remain.
Ms Sturgeon’s message on Thursday night included an image of the words “Europe” and “Scotland” crossing each other with a love heart joining them at the centre.
It was an image that had been projected on to the side of the European Commission building in Brussels last year.
The SNP paid for the stunt, which the Commission said it “had nothing to do with”.
The first minister previously tweeted the image on 31 January when Britain officially left the EU and entered the transition period.
Meanwhile, Scotland’s constitution secretary Mike Russell claimed independence was the “only alternative” – pointing to separate arrangements for Northern Ireland and Gibraltar which allow them to avoid hard borders with the Republic of Ireland and Spain.
Mr Russell said that proposals by the Scotland’s government in December 2016 for it to remain in the single market “could have produced a Scottish deal but were never pursued in EU negotiations” by the UK.
A small number of “Yes for EU” demonstrators gathered outside Holyrood on Thursday to protest against the end of the transition period.