Chavez said missionaries of the New Tribes Mission, based in Sanford, Florida, were no longer welcome. He spoke at a ceremony in a remote Indian village where he presented property titles to several indigenous groups.
"The New Tribes are leaving Venezuela. This is an irreversible decision that I have made," Chavez said on Wednesday.
"We don't want the New Tribes here. Enough colonialism!"
He accused the missionaries of building luxurious camps next to impoverished Indian villages and circumventing Venezuelan customs authorities as they freely flew in and out on private planes.
"These violations of our national sovereignty have to stop," Chavez said.
The group is involved in "true imperialist infiltration, the CIA, they take away sensitive, strategic information", Chavez said, without elaborating.
"And on top of that, exploiting the Indians."
"The group is involved in true imperialist infiltration, the CIA, they take away sensitive, strategic information. And on top of that, exploiting the Indians"
"We don't want to abuse them, we're simply going to give them a period of time (to) pack up their things because they are leaving," Chavez said to applause from hundreds of Indians who sat under tents in Barranco Yopal, the remote village on Venezuela's southern plains.
Nita Zelenak, a New Tribes representative reached by phone, declined to comment on Venezuela's decision or say how many missionaries are working in the country.
The New Tribes Mission specialises in evangelism among indigenous groups in the world's remotest places.
The organisation says it has 3200 workers and operations in 17 nations across Latin America, Southeast Asia and West Africa.
During the ceremony, Chavez granted 15 property titles for more than 665,847 hectares (1.65 million acres) to the Cuiba, Yuaruro, Warao and Karina tribes.
The documents recognise collective ownership of ancestral lands by communities with some 3000 people.
Chavez granted property titles
to various indigenous tribes
"We are doing justice," Chavez said.
"We can now start to say that there is a homeland for the Indians."
Hundreds of people from various indigenous groups attended the ceremony in the southern state of Apure. Chavez also presented keys to tractors, pickup trucks and outboard motors.
"Previously, the indigenous people of Venezuela were removed from our lands. This is historic. It is a joyful day," said Librado Moraleda, a 52-year-old Warao from a remote village in the Orinoco River Delta.
Moraleda received a land title and government pledges of 57 million bolivars ($27,000; 22,000) to build homes and plant cassava and plantains.
'Revolution for poor'
Chavez held the ceremony on the holiday known as Columbus Day, marking the arrival of European explorers in the present-day Bahamas on 12 October 1492.
Chavez's government renamed the holiday Day of Indigenous Resistance three years ago.
Chavez says he is leading a "revolution" for the poor and that defending the rights of Venezuelan's 300,000 indigenous people is a priority.
But poverty remains severe in many Indian communities, and some said they need more help beyond land titles.
"We want the government to help us with hunger, with credit," said Yuaruro Indian Pedro Mendez, 26.
He said his community had asked for an electrical generator and loans to help plant more crops.