Pope Francis said countries breaking away from larger states should be considered on a case-by-case basis.
When asked about the "conflict" between Spain and those in the Catalan region who are campaigning to be a separate state, he said: "All division worries me."
Both the Yes and No camps welcomed the pontiff's comments, with his call for caution pleasing the Better Together camp and his suggestion that "there will be cases that are right and ones that are not" seized on by Yes Scotland.
His observations follow recent comments from President Barack Obama and this week by former US Secretary of State Hillary Clinton, which prompted First Minister Alex Salmond to claim such outside interventions were likely to make "thrawn" Scots more likely to vote for independence.
Mr Salmond did not directly respond to the Pope's comments, leaving it to the Yes Scotland campaign.
Pope Francis, speaking to a Catalonian newspaper, La Vanguardia, also said: "There is independence by emancipation and there is independence by secession. Independence by emancipation include the Americans, who emancipated themselves from European states."
He added that "the independence of people by secession is a dismemberment", citing the "obvious" example of old Yugoslavia involving peoples whose cultures are "so diverse that not even glue could stick them together".
But the pontiff then volunteered: "I wonder if things are quite as clear in the other communities that have been together until now. They should be studied on a case-by-case basis - Scotland, Padania [Northern Italy], Catalonia. There will be cases that are right and ones that are not. But the secession of a people without a precedent of forced unity should be handled very carefully and analysed case by case."
Yes Scotland picked up on the last point, saying: "Scotland's national status has always been recognised by the Catholic Church - indeed, Scotland has been a filia specialis, or favourite daughter, of the Papacy.
"The aspiration to independence is about making Scotland a fairer country - where wealth is shared more equally among all the people who live here, and we banish nuclear weapons once and for all."
Commenting for Better Together, Anne McGuire, the Labour MP for Stirling, said: "This is an interesting and welcome intervention from Pope Francis. The Pope is right to warn about the impact of division on society and to draw the distinction between independence for emancipation and that for secession.
"We live in a large, interdependent world and the best way to secure our future is to work together as part of something bigger.
"Coming on the back of the interventions by President Obama, Hillary Clinton and JK Rowling, the comments from Pope Francis highlight just how important the decision we have to make in September is."
A spokesman for the Catholic Church said: "Although addressing the situation in Catalonia, the Pope's comments are measured and thoughtful and do not seek to prejudge the outcome of any political process."