The police said Dionisio Ribeiro Filho, 59, was shot in the head on Tuesday at the entrance to the Tingua federal reserve, about 30km from Rio de Janeiro city, after he defended it from poachers and illegal palm tree cutters.
"He had received death threats for some time," said Luis Henrique dos Santos, head of the Tingua reserve and a federal employee. "We are working against illegal palm tree cutting and this upset a lot of people."
Filho's death comes after the 12 February killing of prominent US human rights and environmental activist Dorothy Stang as she set up a government peasant farming reserve in the state of Para and defended it from illegal loggers and ranchers.
President Luiz Inacio Lula da Silva launched Brazil's biggest ever crackdown on crime in the Amazon rain forest after Stang's murder caused worldwide outrage.
Stang had received repeated death threats.
Brazil's federal environmental agency IBAMA, which operates the Tingua reserve, has asked for federal police protection for its workers in the 249 square kilometre area of rolling, rain forested hills.
"This business of shutting up ecologists and environmentalists with violence, it's not going to stop," said Edson Bedin, head of IBAMA in Rio de Janeiro state.
Ribeiro was a member of a non-profit environmental organisation that helped set up the park in 1989. He worked to defend it from people harvesting palm trees for heart of palm, a gastronomic delicacy, and trapping tropical birds and animals for illegal sale.
Brazil has already deforested 97% of its Atlantic rainforest, which runs along its coastline and was once a third the size of Brazil's Amazon jungle.