Melbourne Cup contender Cliffsofmoher has been euthanised on track after suffering a fractured shoulder.
French-trained horse Verema had to be put down after an injury suffered in the 2013 Cup.
Fan favourite Red Cadeaux, who finished second on three occasions, was pulled up in the final straight of the 2015 edition of the race and it was later confirmed he suffered a break in his left foreleg.
Limb injuries are the most common cause of death for racehorses on the track. Hind limb injury caused 17 deaths.
Admire Rakti was a Japanese racehorse who collapsed and died from cardiac arrest at the Melbourne Cup in 2014.
In 2013, Verama was euthanised after a leg injury described as horrific by racing experts.
In a statement, RSPCA Australia spokesperson Jane Speechley said: "Our thoughts are with animal lovers across the country who have been affected by this loss, and confronted by the very real risks posed by racing to the horses involved".
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A deluge of rain made for a treacherous start to Melbourne Cup day, forcing delays to races and trains to Flemington Racecourse, and leading to calls for the $7.3 million feature race to be cancelled.
Veterinarians rushed on to the track as soon as the race was completed as a tarp was erected around the fallen stayer.
There was a tragic incident at the Melbourne Cup with The CliffsofMoher breaking down early.
In a tragedy for connections, The Cliffsohmoher suffered a fractured shoulder in the incident and couldn't be saved.
While Cross Counter won the Cup, the Aidan O'Brien-trained horse broke down in the first 600m and failed to run any futher.
An official guides the finishing horses away from the stricken The Cliffsofmoher after the running of the Melbourne Cup on Tuesday.
Described as a "tragedy" by veteran Seven commentator Bruce McAvaney, the United Kingdom raider pulled up lame as the field passed the finishing post for the first time.