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 total of 737 clubs have entered the 2017-18 competition, with 370 of those starting their campaign on the weekend 5-6 August when 185 extra preliminary round ties will be played.
The extra preliminary round is the first of six qualifying rounds before the first round proper on 4 November.
The two Northwich teams will be also face each other in the North West Counties Premier Division next season.
Northwich Victoria were beaten 3-0 by Premier League Sunderland at the Stadium of Light 11 years ago.
There are 18 clubs taking part in the FA Cup for the first time this season, including Whitchurch Alport.
The Shropshire club was formed in 1946 and boasts former Northern Ireland striker Jimmy Quinn among its former players.
Friday's draw took place 41 days after Arsenal beat Chelsea in the 2017 final at Wembley.
All extra preliminary round winners will receive £1,500 in prize money.
Click here for the full FA Cup extra preliminary round draw

The Anglo-Dutch giant said profits on a current cost of supply measure (CCS) - which strips out price fluctuations - jumped to $3.4bn (Â£2.6bn) from $1bn last year.
A 55% rise in oil prices in the first quarter of 2017 compared with a year earlier was the main driver of profits.
Excluding one-off items, profits on the CCS measure were $3.86bn, up 136%.
Shell joins rivals BP, Exxon Mobil, Chevron and Total in reporting better-than-expected results.
More than $1bn in cost savings and budget cuts made over the past three years from cost-cuts and assets sales have also helped to increase cash flow and boost profits.
But Shell chief executive Ben van Beurden said the company would be making $25bn worth of investments this year in new oil and gas projects. "We continue to reshape Shell's portfolio and to transform the company," he said in a statement.
Oil and gas production, known as upstream, rose 2% in the quarter to 3.752 million barrels of oil equivalent from 3.905 million in the fourth quarter of 2016. A number of new fields continued to ramp up output, particularly in Brazil and Kazakhstan.
Earnings from refining, marketing and chemicals also rose 20% to $2.5bn.
"We saw notable improvements in upstream and chemicals, which benefited from improved operational performance and better market conditions," Mr van Beurden said.
Investors welcomed the results, marking Shell's shares up 3% at the start of trading.
Energy producers across the world are reaping the benefits of higher oil prices, which have strongly increased their revenues and profits.
Crude has recovered thanks to the Opec oil producers' cartel adhering to a production cut agreed late last year. However, the oil price has eased back recently.
A train made a return journey from London Victoria to Gatwick Airport without passengers in a dispute over a new service.
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Northwich Victoria, who reached the second round two seasons ago and the third round in 2006, will face local rivals 1874 Northwich - a breakaway club formed five years ago - in the extra preliminary round of the FA Cup.
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Royal Dutch Shell's profits surged in the first three months of the year on the back of rising oil prices.