Hua Zui Ba, 20, and Bing Xing, 23, have lived in Madrid since 2007 and in the past decade have had three offspring: Chulina, 7, and twins You You and Jiu Jiu, 2.
They will be replaced with a new panda couple sent by the China Wildlife Conservation Association, zoo authorities said.
China has loaned its treasured bears to zoos in various countries over the years as goodwill animal ambassadors while also running conservation programmes back home that has improved their status from endangered to vulnerable.
Spain has been part of that programme for more than 40 years, hosting Chu Lin, the first panda born in captivity in Europe, for 14 years at Madrid Zoo in the 80s and 90s.
On Thursday morning, the family were packed up in specially-built cages they have been adapting to over months as part of their daily routine.
Each panda travels its own reinforced stainless steel cage, personalised with their name and picture.
The five-panda family's travel kit includes 100kg of bamboo (220 lbs), 60 litres (16 gallons) of water and several kilos of apples, their favourite fruit.
The bears were taken to the airport in two, 22-metre articulated lorries under police escort.
Then they set off on an 11-hour direct Air China flight to Chengdu with a veterinarian from Chengdu and two of their keepers, who will stay on to ensure they adapt well to their new home.
The population of giant pandas in the wild has grown from around 1,100 in the 1980s to 1,900 in 2023 and there are 728 pandas in zoos and breeding centres around the world.
(Reporting by Marco Trujillo, Violeta Santos Moura and Emma Pinedo; editing by Inti Landauro, Aislinn Laing and Sharon Singleton)
