The University of Glasgow and the University of Aberdeen, two of Scotland's ancient universities, have resigned after the CBI formally registered as a supporter of Better Together.
Aberdeen's Robert Gordon University has also expressed its disapproval at the decision, but has decided to remain a CBI member.
Dundee University is reviewing its membership at a meeting later this week, a spokesman said.
Scottish Enterprise, VisitScotland, STV, the Balhousie Care Group and energy company Aquamarine Power have all resigned from the CBI.
A Glasgow University spokesman 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. We believe that the role of a university is to inform the public debate through the contribution of its academic and research expertise.
"Whatever the people of Scotland decide in this autumn's referendum, the university will continue its ambitious mission to be counted among the world's top institutions of learning and research."
A Robert Gordon University (RGU) spokesman said: "The purpose of the university's membership of the CBI is to assist in developing links with potential business partners. Such memberships are reviewed annually by the principal, in discussion with senior university officers.
"The university is strictly neutral in relation to the independence referendum, and does not approve of the CBI statement. However, we are not in the CBI in order to address Scottish independence.
"Within RGU, as in other organisations, there are individuals with strong views on either side of the independence debate. The university itself will remain neutral."
CBI director general John Cridland defended the decision to back Better Together this morning, insisting it was made for "for compliance reasons" and did not represent any wish to campaign to influence voters' opinions.
It emerged on Friday that CBI Scotland had registered with the Electoral Commission, meaning it can spend more than £10,000 on campaigning during the referendum period.
Registering as a campaigner also gives access to the electoral register and the right for representatives to attend postal vote opening sessions, polling stations and the counting of votes.
Mr Cridland told BBC Radio 4's Today: "We are not trying to campaign to influence the Scottish voter but we are a business organisation and on the business issues - jobs in Scotland, growth in Scotland, living standards in Scotland - we have a view: we don't think the economic case for independence has been made and we think the economy in Scotland and the economy of the United Kingdom is stronger together."
He added: "I regret any CBI member leaving. That is a matter of considerable regret to me as chief executive. But I respect the fact that there are a variety of views.
"Nothing changed this weekend about the CBI's position on the issue. All that changed is that for compliance reasons, we decided that we needed to register to be on the right side of those regulations."
Balhousie chairman Tony Banks said CBI took the decision without consulting its members and has challenged the organisation to produce evidence that it has followed due democratic process.
Mr Banks, who chairs the pro-independence campaign Business For Scotland but insists his business is "neutral" in the independence debate, has predicted there will be more walkouts in the days ahead.
Mr Banks is "unwilling to fund" the £150,000 the CBI will be permitted to spend campaigning against independence, he said.