Aslef said a proposed resolution to the dispute was rejected by 54.1 per cent in a turnout of 72 per cent.
General secretary Mick Whelan said: “We understand and support the decision arrived at democratically by our members and will now work to deliver a resolution in line with their expectations.”
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Aslef said 953 ballot papers were sent out, with 693 returned. There were 317 votes in favour (45.9 per cent) of the proposed deal, and 374 against (54.1 per cent), with two invalid papers.
The union’s leaders had agreed the deal during 11 days of talks held at the headquarters of the TUC, which ended earlier this month.
Members of the Rail, Maritime and Transport union are also embroiled in a bitter row with Southern over staffing and are set to stage a 24-hour strike on February 22.
Nick Brown, Chief Operating Officer of Southern’s owners, Govia Thameslink Railway, said: “Naturally we’re saddened and hugely disappointed, as will be our passengers, with today’s decision by drivers, particularly as the agreement carried the full support and recommendation of the Aslef leadership.
“We now need to understand the issues which led to this outcome and we’ll be seeking to meet with the union as soon as possible to see how we can agree a way forward.”
The RMT attacked the proposed agreement worked out between Aslef’s leaders and the company, describing it as a “shocking betrayal” of workers and passengers.
Aslef’s leaders said the deal had been misunderstood, insisting it would lead to safety improvements.
The two unions have been in dispute with Southern for almost a year over staffing issues, including whether a second, safety critical member of staff should be guaranteed on trains.
A series of strikes have been held, causing chaos for Southern’s 300,000 passengers, who now face the prospect of further disruption.
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RMT leader Mick Cash said: “This ballot was entirely a matter for Aslef and their Southern members. RMT has remained focused on the industrial and public campaign to protect the safety of the travelling public and to put access and safe operation before profits.
“RMT will now look to take that campaign into its next phase working with our sister rail unions, the wider trade union movement and the passengers who use the railway.
“RMT repeats the call to Southern to give the guarantee of a second, safety critical member of staff on their trains and to sit down with the unions in new talks around the issue of safe train despatch.”
David Sidebottom, of independent watchdog Transport Focus, said: “The hope that services would improve on Southern has now been dashed for their passengers.
“They have had enough of the on-going industrial action. They have faced months of lost time, lost money and deep frustration at not being able to rely on the trains.
“It is vital that all parties in this dispute get back around the table to bring the services back to normal as soon as possible.”
Andy McDonald, shadow transport secretary, said: “Southern services are abysmal even without strike action, but the failure to resolve this industrial dispute is bad news for both staff and passengers.
“The Government and Govia Thameslink Railway have failed to guarantee passenger safety or accessibility for disabled passengers who face a loss of independence with the expansion of driver-only operation services.
“For the sake of long suffering passengers, all parties need to get back around the negotiation table and thrash out a deal that delivers a safe, reliable and accessible service.”