In a speech to Latin American and Caribbean bishops at the end of a visit to Brazil, the Pope said the Church had not imposed itself on the indigenous peoples of the Americas.
They had welcomed the arrival of European priests at the time of the conquest as they were "silently longing" for Christianity, he said.
Millions of tribal Indians are believed to have died as a result of European colonization backed by the Church since Columbus landed in the Americas in 1492, through slaughter, disease or enslavement.
Many Indians today struggle for survival, stripped of their traditional ways of life and excluded from society.
"It's arrogant and disrespectful to consider our cultural heritage secondary to theirs," said Jecinaldo Satere Mawe, chief coordinator of the Amazon Indian group Coiab.
Several Indian groups sent a letter to the Pope last week asking for his support in defending their ancestral lands and culture. They said the Indians had suffered a "process of genocide" since the first European colonizers had arrived.
Priests blessed conquistadors as they waged war on the indigenous peoples, although some later defended them and many today are the most vociferous allies of Indians.
"The state used the Church to do the dirty work in colonizing the Indians but they already asked forgiveness for that ... so is the Pope taking back the Church's word?" said Dionito Jose de Souza a leader of the Makuxi tribe in northern Roraima state.
Pope John Paul spoke in 1992 of mistakes in the evangelization of native peoples of the Americas.
Pope Benedict not only upset many Indians but also Catholic priests who have joined their struggle, said Sandro Tuxa, who heads the movement of northeastern tribes.
"We repudiate the Pope's comments," Tuxa said. "To say the cultural decimation of our people represents a purification is offensive, and frankly, frightening.
"I think (the Pope) has been poorly advised."
Even the Catholic Church's own Indian advocacy group in Brazil, known as Cimi, distanced itself from the Pope.
"The Pope doesn't understand the reality of the Indians here, his statement was wrong and indefensible," Cimi advisor Father Paulo Suess told Reuters. "I too was upset."