Hull City's crowd pilot on Saturday has been cancelled amid a rising number of Covid-19 cases in the city.
The League One game against Crewe was one of eight in the English Football League allowing supporters to attend.
But with infection rates in Hull more than tripling in the past week, the local council has cancelled the government-led pilot.
Up to 1,000 fans will still be allowed at the seven other games and they will be the first EFL crowds in six months.
All games in the Championship, League One and League Two have been behind closed doors since March, although 872 fans attended an EFL Trophy match at Cambridge United earlier this month.
Hull had already indicated they would only invite 600 supporters on Saturday "to manage risk" before Thursday's announcement that the pilot was no longer taking place.
"In order for the proposed 'return of fans' pilot match to go ahead at the Hull City v Crewe Alexandra game at the KCOM Stadium this Saturday, it was necessary to meet 10 public event assessment criteria, established by the Association of Directors of Public Health," a joint statement from Hull City Council and the head of stadium operations said.
"The first of these states that there can be no significant increase in the area's coronavirus infection rate ahead of an event. Unfortunately, the rate in Hull currently stands at 15.3 infections per 100,000 people, up from 4.2 per 100,000 people last week.
"Consequently, on the grounds of public health and public safety, this pilot cannot go ahead and the game will have to be played without fans in attendance.
"We appreciate this decision will be disappointing for many but, in the current circumstances, it is the safest and most appropriate decision and has been made in the best interests of our residents and our visitors."
The remaining seven matches, all taking place on Saturday, are set to have up to 1,000 fans each (all kick-off times 15:00 BST unless stated):
Middlesbrough v AFC Bournemouth
Norwich City v Preston North End
Blackpool v Swindon Town
Charlton Athletic v Doncaster Rovers
Shrewsbury Town v Northampton Town
Carlisle United v Southend United (13:00 BST)
Forest Green Rovers v Bradford City