The Confederation of British Industry (CBI) Scotland represents many businesses across the country, and has already stated its support for the pro-union campaign.
Organisations or individuals must register with the Electoral Commission if they want to 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.
A CBI spokesman said: "The CBI has clearly stated its position in the Scottish referendum debate, that Scotland and the rest of the UK are stronger together as part of the union
"We have registered this with the Electoral Commission in accordance with the law."
CBI director Iain McMillan gave his views on the implications of independence when he gave evidence to Holyrood's Economy Committee earlier this month.
He told MSPs that key issues for members include currency, EU membership, the deficit and what he called the ''fragmentation'' of the UK market.
''In terms of an independent Scotland, there would be every bit as much need to attack the deficit and deal with fiscal consolidation going forward and that of course would result in many difficult decisions about tax and spend,'' he said.
''This would not be a land of milk and honey. It would be extremely difficult with many painful decisions to be taken."
But Tony Banks, chairman of the pro-independence group Business for Scotland, said he will now quit the CBI.
Mr Banks, who is also chairman of the Balhousie Care Group, said: "It is abundantly clear that the CBI is not representing its members' views honestly. I am therefore writing to the director-general, John Cridland, today, withdrawing my company membership."
He said the CBI has attempted to "hijack its members without proper consultation" on their views and "many must now feel they are in an impossible position".
He also questioned whether the CBI is being open about how many members it has.
Business for Scotland has 1,700 members, he said.
Chief executive of Business for Scotland Gordon MacIntyre-Kemp said: "We have been calling in public and requesting in private that the Electoral Commission (Scotland) ask the CBI to register as a No campaigner.
"That they did so on Thursday is a major victory for us and a blow to the No campaign. We have always accepted Business for Scotland will register as a campaign participant once the CBI has and we will now do so now."
A second firm also said it will leave the CBI. Martin McAdam, chief executive officer of wave energy company Aquamarine Power, said: "The CBI has registered with the Electoral Commission as a No campaign backer without consultation with its members.
"As a business, Aquamarine Power has been firmly neutral on the matter of independence. We have adopted this view after consultation with our board and management team and as a consequence we can no longer remain members of the CBI.
"Although Aquamarine Power's staff and board members may have personal views on the matter of independence, this has no influence on our agreed company position."
Electrical contractors union Select, which represents 1,250 companies with 18,500 employees, has demanded the CBI review its decision.
While there has been no threat to leave the CBI, the union said the Better Together affiliation does not reflect its views.
Select said: "The owners of our member companies and their employees, like the rest of the Scottish population, cover the full range of political affiliations and, within Select, we are agreed that the way in which each and every person associated with Select votes on September 18 is a matter for them alone.
"We would appreciate it if CBI reviewed this decision before taking action."