The broadcaster has joined the Balhousie Care Group and wave energy company Aquamarine Power in resigning from the Confederation of British Industry (CBI) Scotland.
STV said it had a duty of impartiality and was left with "no choice but to resign" from the confederation.
It emerged yesterday that the CBI, which represents many businesses across the country, 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.
The confederation has faced criticism that its position does not accurately reflect its members' views, and it failed to consult them before formally registering to campaign for the union.
A CBI spokesman said that Scotland and the rest of the UK are "stronger together as part of the union".
Broadcasters such as STV and the BBC are bound by strict rules when it comes to reporting in the final campaigning period before elections and referendums.
A statement on the STV News website said: "STV is a public service broadcaster with a duty of impartiality and as such we have no corporate or editorial position on the independence referendum in September.
In light of CBI Scotland's decision to register with the Electoral Commission we have no choice but to resign our membership of CBI Scotland forthwith."
Tony Banks, chairman of the Balhousie Care Group, quickly responded to the CBI's position yesterday and said his business will now leave the confederation.
Mr Banks, who is also chairman of the pro-independence group Business for Scotland, 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.''
Martin McAdam, chief executive officer of Aquamarine Power, said there had been no consultation before the CBI decision.
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.