A spokesman for People for the Ethical Treatment of Animals said: "Damien Hirst's quest to be edgy is as boring as it is callous."
"It does not matter whether Hirst killed the animals himself or sat by while thousands of them were massacred for his own unjustifiable amusement.
"Butterflies are beautiful parts of nature and should be enjoyed in the wild instead of destroyed for something predictable and unimaginative."
An RSPCA spokesman added: "In this exhibition, butterflies are forced to exist in the artificial environment of a closed room for their entire lives.
"There would be national outcry if it involved any other animal, such as a dog.
"Just because it is butterflies, that does not mean they do not deserve to be treated with kindness."
Dr Martin Warren, chief executive of Butterfly Conservation, said: "It is very sad to hear of the death of so many butterflies.
"We are very concerned that this work represents a throwaway approach to living creatures and encourages a lack of respect for the environment."
The artwork, shown at the Tate Modern gallery in London between April and September, was part of a retrospective of Hirst's work, which attracted almost half a million visitors.
It used tropical butterflies from the Owl and Heliconius species, which can live for up to nine months in their natural habitat.
Those used in the exhibition survived for between a few hours and several days, suggesting their lifespan was shortened.
Bowls of fruit, flowers and sugared water were left in the rooms to allow the butterflies to feed.
A spokesman for Tate Modern said: "The butterflies used in this work were all... selected from varieties known to thrive in the conditions created.
"The butterflies lived out the final stage of their natural life cycle inside this room. Around 400 butterflies were introduced to the exhibition over the course of each week."
In a statement, Hirst said: "A butterfly expert was employed at considerable cost.
"Perfect living conditions were replicated and this resulted in many butterflies enjoying longer lifespans due to the high quality of the environment and food provided."
