Train drivers' union Aslef said passengers were forced to leave the train due to a row over running longer, 12-car trains without a guard.
The union is now balloting drivers over industrial action.
Gatwick Express said it had been talking to Aslef for a year and was "puzzled" by the situation.
Trains have been introduced gradually since February, with 12-car trains due to officially replace 10-car services from Monday.
Gatwick passenger services director Angie Doll said a driver was not prepared to carry passengers on Saturday's first 12-car service.
She said: "Unfortunately the train was cancelled and around 30 passengers were moved to a train on the adjacent platform which left seven minutes later.
"The driver did take the train to Gatwick and back but not with any passengers on board."
The company said letting the train out of Victoria without anyone on board was the best solution.
It said the train would otherwise have blocked the busy platform and caused significant disruption, with no time available to split it or take it elsewhere.
Ms Doll said the union was aware the trains were being introduced and drivers had already operated the trains and been given training.
She said the company was open to further talks.
Simon Weller, from Aslef, said the union had told Gatwick Express that members were not prepared to work 12-car trains.
He said: "The problem is running driver-only trains with passengers.
"The problem we have is with passengers boarding and alighting with only one person on the train.
He said the union wanted Gatwick Express to put guards on the trains and said: "The driver-only operation is a legacy of the shoestring economics of British Rail."
A train made a return journey from London Victoria to Gatwick Airport without passengers in a dispute over a new service.