By Alex MacDonald 

LONDON--More than 130 business leaders have signed an open letter slamming the case for Scottish independence, marking the biggest intervention by the U.K. business community in the referendum debate so far.

Voters will decide on Sept. 18 whether Scotland should exit the U.K., ending a union agreement with England that has been in place since 1707. Unionists and secessionists have been sparring over who can offer Scots the brightest economic future.

Polls suggest Scots will choose to remain in the U.K. when they cast their ballots, but pro-independence campaigners believe a swath of undecided voters could still tip the balance in their favor.

In the open letter, published in the Scotsman newspaper Wednesday, the group of top businessmen said that after looking carefully at the arguments on both sides of the debate they concluded that "the business case for independence has not been made."

The group includes signatories such as Andrew Mackenzie, chief executive of the world's largest diversified miner BHP Billiton Ltd, Douglas Flint, chairman of HSBC Holdings PLC and Simon Thomson, CEO of Cairn Energy PLC.

"Uncertainty surrounds a number of vital issues including currency, regulation, tax, pensions, EU membership and support for our exports around the world; and uncertainty is bad for business," the group wrote in the letter, adding the outcome of the referendum would affect generations to come.

The letter follows cautious comments on the referendum from a number of executives.

Royal Bank of Scotland Group Chairman Philip Hampton has previously said the vote has caused "a great deal of uncertainty" and that the bank is considering the possible business implications of a yes vote and its response.

While Ivan Menezes, the CEO of Diageo PLC, the world's biggest producer of Scotch whisky, has said it is "extremely important" for the company--and the Scotch whisky industry--to remain part of the European Union so it could benefit from "free-trade agreements around the world."

An independent Scotland's immediate membership in the EU has been cast into doubt because it would require the approval of all existing member states, often a drawn-out process.

Write to Alex MacDonald at alex.macdonald@wsj.com

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