The universities of Glasgow, Aberdeen and Edinburgh became the latest members to quit the business lobbying body in a development seized on by the pro-independence side. The institutions - which rely heavily on public funding - said the CBI's decision to register as a campaign group with the Electoral Commission was at odds with their political neutrality. They followed two Scottish Government agencies, Scottish Enterprise and VisitScotland, in resigning.
Broadcaster STV and Balhousie Care Group - a company chaired by businessman and prominent Yes campaigner Tony Banks - have also quit in recent days, while a fourth university, Glasgow Caledonian, is to consider its membership today.
Aberdeen's Robert Gordon University yesterday expressed its disapproval at the decision but decided to remain a CBI member.
In a statement, a spokesman for the University of Glasgow said: "As an institution we have a strictly neutral position on the independence debate. We have carefully considered the decision of CBI Scotland to register with the Electoral Commission and decided that in order to maintain our impartiality we must resign our membership forthwith."
An Aberdeen University spokesman said: "In light of the position taken by CBI Scotland, the University of Aberdeen feels it is inappropriate to continue our membership of this body.
"The University of Aberdeen does not take an institutional position on the constitutional future of Scotland."
Confirming its decision, a University of Edinburgh spokesman said: "While the University of Edinburgh hosts debate from all sides in the discussion around Scottish independence, we have a strictly neutral position on the issue as an institution."
Robert Gordon University "is strictly neutral in relation to the independence referendum, and does not approve of the CBI statement", its spokesman said.
He added: "However, we are not in the CBI to address Scottish independence."
Though independent institutions, universities receive around a third of their income from Scottish Government grants distributed by the Scottish Funding Council. In addition they rely on research grants and student fees paid by the Scottish Government.
The Scottish Government has already backed the decision of its tourism agency VisitScotland and economic development body Scottish Enterprise to resign, saying it was "clearly inappropriate for government agencies to remain in membership of CBI".
John Cridland, the director general of the CBI, said: "I am very sad to lose Scottish university members. Education of our young people is vitally important to everyone in business, and I have greatly valued the contribution that Scottish universities have made to the CBI.
"We are all in uncharted waters and recognise and respect that difficult decisions may need to be taken.
"We have a clearly stated position that Scotland and the rest of the UK are stronger together on economic grounds as part of the Union and this reflects the views of the vast majority of our members."
Earlier, he insisted his organisation's decision to register as a No supporter was made "for compliance reasons" and did not represent any wish to campaign to influence voters' opinions.
The decision by CBI Scotland means it can spend up to £150,000 on campaigning.
Business for Scotland chief executive Gordon MacIntyre-Kemp said: "The CBI must come clean on what basis it has developed its No campaign position against Scottish independence. The Scottish people have a right and the CBI has an obligation to explain to its members how it reached the conclusion to take a landmark political stand against independence."
Mr Cridland told BBC Radio 4's Today: "I regret any CBI member leaving."
A CBI Scotland source said: "We have received a large number of emails of support from company chief executives over the weekend which has been really heartening but no-one has wanted to put anything into the public domain."