Members of train drivers' union Aslef have rejected the deal their union and Southern Rail had agreed.
Earlier this month, a TUC-brokered deal was agreed in the long-running dispute between Southern rail and Aslef over the role of the guard, in the wake of fresh talks.
It was expected to bring the row over the so-called driver-only operated trains to a close and end the ongoing industrial action that had disrupted the network, subject to the referendum of Aslef members.
But members have rejected the proposed deal with 54.1 per cent voting no on a turnout of 72.7 per cent.
Mick Whelan, general secretary of Aslef 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 union said it will now seek to "re-open negotiations" with Southern to resolve the outsanding issues in "a way which works for drivers, passengers and the company".
Nick Brown, chief operating officer of Govia Thameslink Railway (GTR), Southern’s parent company, 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 deal had sparked criticism from the Rail, Maritime and Transport (RMT) union too, which had called it a "shocking betrayal" of workers and passengers after seeing details of the deal. The RMT said the agreement would lead to de-staffing of trains.
Southern rail services have been hit by months of disruption as Aslef and the RMT carried out numerous walkouts over the role of the guard.
Aslef suspended strikes planned for last month after Southern agreed to fresh talks at the Trades Union Congress (TUC).
The RMT's dispute with Southern remains unresolved, after new talks broke down on Tuesday. The union has since called for a fresh 24-hour walkout next week on Wednesday 22 February
RMT's general secretary Mick Cash said "the abject failure" of Southern rail to take "the safety issues seriously has left us with no option but to confirm further action".
He added: "The full detail of Southern’s plan is far worse than anyone could have anticipated. This is dire news, not just for staff, but for passengers who rightly demand a safe, reliable and accessible service. RMT will not walk away from the fight for a railway that puts public safety before private profit."
Nick Brown, chief operating officer of Govia Thameslink Railway (GTR), Southern's parent company, said: "We are disappointed that the RMT is going to heap yet further misery and disruption on the travelling public. We aim to run as full a service as we can."