It claims that up to 2.7 million chickens suffered as a consequence of the Oxford University research, which was designed to measure the survival rates of chickens packed together at levels far exceeding currently accepted industry limits.
The charity says that the research goes against the recommendations set by the Government's own advisory body, the Farm Animal Welfare Society.
The RSPCA is concerned that the research has been timed to coincide with upcoming European legislation covering the welfare of broiler chickens.
It believes that the results of the research, which concluded that heat and humidity rather than the density of the birds were the main causes of death, may be used to try to increase the current limits on stock levels.
It is also demanding that Defra explain why it inflicted suffering on the chickens for the research, and will appeal to European legislators to ignore the results.
The RSPCA also attacked the department for failing to apply for a Home Office licence that would have enabled the research to have been observed by its inspectors.
"Government funded the work so it can't distance itself from the implications," said Julia Wrathall, head of the RSPCA's farm animal department. "Defra was unusually secretive, and now we know why."