Lucia Newman, Al Jazeera's correspondent in Santa Cruz, said government supporters blocked roads to prevent people from reaching polling stations.
"They have burned ballot boxes and in some areas there have been various serious clashes between pro and anti-government groups," she said.
"The turnout has been pretty high but these kinds of violent incidents are marring the process."
The move towards self-rule in Santa Cruz, which holds important gas resources, is expected to gain significant backing.
Surveys have suggested that up to 70 per cent of the region's 900,000 voters could support the move.
Local authorities in the eastern region are expected to pass laws giving them greater power over the province's finances and security operations if the referendum is passed.
Newman said that the Santa Cruz vote was just the beginning of a series of ballots on autonomy in Bolivian regions.
"After this vote, there will be three other provinces that will follow suit. There is going to be a domino effect - a large part of Bolivia is going to be voting for autonomy," she said, speaking in Santa Cruz.
"While [the vote] may technically not be constitutional, there is a certain political legitimacy because it comes from a democratic vote.
|Surveys suggest that 70 per cent of Santa Cruz |
voters will support greater autonomy [EPA]
"This is going to force the government to negotiate. Both sides need each other and, despite what the government is saying, neither the state nor the provinces want to break away. There is an impasse here."
Mario Ayala Ferrufino, permanent secretary of the Supreme National Defence Council, said on Saturday that the vote was a threat to the territorial integrity of Bolivia.