Chievo Verona, who spent 17 seasons in Serie A, were wound up after failing to find an investor to resolve their financial problems, the local authorities announced on Tuesday.
Chievo were in Serie B last season but failed to satisfy the Italian second division's financial solvency criteria and were relegated to Serie D.
They needed to put their affairs in order to enter that competition before it kicked off its season.
The deadline for Serie D registration was Tuesday and the club desperately sought a buyer to bail it out but failed.
"The deadline for registering for the championship, which is about to begin, passed at midday," announced the Verona town hall.
The club's former captain Sergio Pellissier, who played almost 500 games for them, tried to put together a rescue plan, but signalled his failure on Instagram on Saturday.
"This is one of the saddest days of my life," he wrote. "I tried with all my strength and I really hoped to succeed in starting again and continuing this fantastic story."
"I regret that I didn't have enough time, it's a really complicated period to be able to find sponsors," he said.
They won promotion to Serie A for the first time in 2001.
Nicknamed the "Flying Donkeys", and operating on a shoestring budget, they survived until 2019.
They even finished fourth in 2006 to qualify for the Champions League after AC Milan, Fiorentina and Lazio were each docked 30 points for their involvement in the Calciopoli scandal. They were knocked out in a qualifying round by Levski Sofia.
Chievo finished eighth in Serie B last season.