ASLEF drivers voted against an agreement that would have ended a bitter dispute with the franchise.
The union had argued with Govia Thameslink Rail (GTR), Southern’s parent company, over plans to run driver-only trains, which they claimed posed a safety risk.
Its members voted 54.1 per cent to 45.9 per cent against the deal and the turnout was 72.7 per cent.
ASLEF drivers first went out on strike in December over the issue following a break-down in negotiations with the company.
Nick Brown, chief operating officer of GTR, 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.”
Mick Whelan, general secretary of ASLEF, the train drivers’ union, 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."
The Rail, Maritime and Transport union is still in dispute with Southern over changes to guard’s contracts that would hand responsibility for opening and closing carriage doors to drivers.