The performance, named Moses and Aaron, is due to run at Madrid's Teatro Real from My 24 to June 17 and includes a scene featuring the bull and a naked woman.
Petition organisers are calling for Spain's Ministry of Culture to ban the show, or force producers to replace the live bull with a statue.
The petition reads: 'The inclusion of the animal involves exposure to light and loud sounds, constant transport and unnecessary stress' during the scene it features in and the show.
'The use of animals in any kind of entertainment is humiliating, cruel and unnecessary, therefore we ask the Minister of Culture, Inigo Mendez de Vigo, and the Mayor of Madrid, Manuela Carmena, to cancel the work or use a statue instead of a living being.'
Campaigners also said they suspected the animal was drugged 'to be able to better control the scene, which concerns a 1,500kg animal'.
However, the theatre has stated the bull takes no painkillers or drugs and has two carers on hand to ensure its well being, The Local reported.
To date, the appeal has received almost 45,000 signatures.
According to El Pais, the bull's owner earns about 5,000 euros (£4,000) for each show in which his animal plays the role of the Golden Calf.
And a previous run of the opera in Paris was not without controversy - cast members complained their animal colleague earned more per show than he did in a month.
Animal rights groups also protested its inclusion in the Paris show, with a petition containing 30,000 signatures sent to the culture ministry opposing it, the paper reported.
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