Mario Cossio, the mayor of Tarija, said "this day will mark a new page in our national history" that would clear the way for a "better future".
But Morales has said he will not recognise the results of the Tarija vote which he branded as illegal and a waste of resources.
Morales has been pushing his wealth-distribution agenda, saying it will benefit the poorer, mostly indigenous Bolivians of the mountainous western regions.
|Tarija has joined Santa Cruz, Beni and
Pando in voting for autonomy
Tarija has only 400,000 inhabitants but has 85 per cent of the country's natural gas, which accounts for 13 per cent of Bolivia's gross domestic product.
The president cancelled a visit to Tarija last week "for security reasons", officials said, and on Saturday, a dynamite explosion damaged a television station in Yacuiba, home to strong opposition to the referendum.
And on Sunday pro- and anti-Morales demonstrators set up competing barricades hundreds of metres from each other in Tarija city, blocking the main road north to Potosi and the capital La Paz.
The referendum also recorded high abstention rates in townships where pro-government, anti-autonomy sentiments were strong.
Political dialogue has collapsed between the Morales-led central government and the opposition, led by the Podemos party and the pro-autonomy governors.
Three other provinces – Santa Cruz, Beni and Pando – are also resisting Morales's policies to redistribute land and resources.
To counter the movement, Morales has announced his own referendum for August 10, on his government and on provincial governors.