Nicola Sturgeon has called on Prime Minister Theresa May to "do the humane thing" by guaranteeing EU nationals living in the UK the right to remain.
The First Minister said the prospect of deportations as a result of the Brexit vote "strikes at the heart of humanity" and urged the UK Government to end the uncertainty faced by three million people.
Sturgeon addressed the issue at a special Scottish Cabinet meeting in Edinburgh featuring an audience of EU nationals living in Scotland.
The First Minister told them it "breaks her heart" that she is unable to guarantee their futures because immigration policy is reserved to Westminster.
So far, the UK Government has refused to rule out the possibility of future deportations despite making repeated moves to reassure concerned EU nationals after June's vote to leave the European Union.
One Italian audience member, Caroline Magoha, spoke of uncertainty over her 13-year-old son's education, adding he had been bullied at school in the build-up to the EU referendum.
Urging politicians not to make EU nationals from the UK the new "refugees of Europe", Ms Magoha said: "We have to live with our bags half-packed, our feet halfway out of the door.
"I don't have any trust whatsoever in the Westminster government. They will wake up one day and say Article 50, EU members, you have to be out within six months. They are capable of that.
"It is inhumane. It is against the basic human rights of children. Brexit is ruining the future of Scottish children."
The First Minister used the event to put pressure on the Prime Minister to clarify the UK Government's stance on the issue, describing her frustration at having no say on immigration issues.
She said: "It really breaks my heart that as First Minster, as the elected leader of this country, I am not able to sit here and give you the guarantees and the certainty that you want.
"This is the one that strikes at the heart of humanity. People living here and trying to get on with their lives here should not suddenly have this question mark over their future, and the UK Government could at the stroke of a pen, today if it chose to, put an end to this uncertainty.
"End this uncertainty, end it now, do the humane thing."
Sturgeon added: "I think it is unthinkable that people living here would be asked to leave. I think the uproar that that would cause, rightly, would be immense.
"But the fact remains that until that commitment is given, people are going to worry and have that uncertainty."
Sturgeon's answers were repeatedly me with applause from the audience but others accused the First Minister of stoking tensions between the Scottish and UK governments.
Patrick Harkness, from Northern Ireland, said while Ms Sturgeon had sought to reassure EU nationals, she was building her own "cold house" while nationalists were "sneering at British people".
He said: "Your backbenchers openly talk about how much people may or may not hate the English and you do not admonish them. Now you're misrepresenting my Remain vote to further yet more separatism.
"First Minister, I think we should recognise that you are building a cold house, too. When are you going to hold an event such as this for UK citizens to reassure us that your nationalists will no longer speak division and hatred against us?"
Sturgeon responded by saying: "I admonish anybody who is anti-anybody on the basis of their nationality, whatever that nationality may be.
"My belief in Scottish independence never has been and never will be based on a sense of where people come from."
She then went on to point out that although she could have called a second independence referendum in the immediate aftermath of the Brexit vote, she had instead chosen to look at all the options available to secure Scotland's place in the EU.
A UK Government spokesman said: "The Prime Minister has been clear that she wants to protect the status of EU nationals already living here and the only circumstances in which that wouldn't be possible is if British citizens' rights in European member states were not protected in return.
"We are consulting with businesses and other stakeholders across Scotland about the impact of leaving the EU.
"Those discussions are covering a range of issues, including freedom of movement.
"We are determined to take a 'team UK' approach to leaving the EU, working closely with the Scottish Government and other partners as we form our negotiating strategy, and we will look at any suggestions put forward."