Concerns were raised after the death of a horse, called Periclea, during trials for this year's Palio on Monday.
She fell and broke her front right foot during an early heat for the first race, which will be held on July 2nd.
The seven-year-old filly fell at the first corner of the San Martino curve after tripping over the legs of the horse in front, La Stampa reported. It was set to be Periclea's first Palio, but the mare had already competed in similar events including the Palio di Ferrara in May.
The animal was given immediate treatment and taken to a local veterinary clinic – but her injuries, which included a broken foot, were too severe for her to be saved.
"This is the umpteenth death that could have been avoided by stopping this shameful and bloody event," a member of animal rights group, Partito Animalista Europeo, told La Stampa.
"Our legal office have sent a cease and desist order to the prefect of Siena as well as to interior minister Angelino Alfano to call for the suspension of the event."
It is unlikely their attempts to stop the race, which started in its modern form in 1656, will be successful.
However, according to the Anti-Vivisection League, 48 horses died at the Palio between 1970 and 2007. More stringent laws regarding safety were brought in the 1990s, including alcohol tests for jockeys, but horses continue to die during the event.
The famous race is held twice a years on July 2nd and August 16th, and lasts about 90 seconds. Riding bareback, the jockeys circle the Piazza del Campo three times and are frequently thrown from their horses.
During the Palio of August 16th 2004 the horse running for the ward of Bruco fell and was trampled to death, which brought complaints from animal rights groups as well as concerns for the safety of the riders.
For the occasion of the Palio, the Piazza is filled with turf and dirt in order to form a racetrack. It is a hugely popular event among both locals and tourists and draws crowds of 50,000 people.
The race has deep traditions in Siena, but is fiercely competed by neighbouring districts of the city, which often employ underhand tactics, such as bribery and doping, to try and get the upper hand.
During the race the jockeys shove, whip and distract their fiercest rivals to try and stop them from gaining the coveted prize, which is an embroidered silk banner known as a Palio, from which the race derives its name.