Police charged twice in order to move protesters away from gates to the work site and a number of people were injured.
Some of the protesters suffered bruising and others felt ill after the clashes, union sources said.
The removal of the olive trees was suspended, but later resumed.
Some 61 trees had been removed by the end of the day with about 150 left to go, local officials said.
Tension remained high late in the day and demonstrators threw stones at police as trucks entered the site, injuring two officers.
The police baton-charged the protesters again to stop them entering the site.
Mayors and councillors from 'No TAP' towns and villages took part in the protest, as did two regional councillors, Cosimo Borracino of the Italian Left (SI) and Antonio Trevisi of the anti-establishment 5-Star Movement (M5S), along with M5S MP Daniela Donno.
The Trans Adriatic Pipeline, which was recently approved by the environment ministry and by the Council of State, Italy's highest administrative court, aims to bring Caspian gas to Europe.
Puglia Governor Michele Emiliano said the regional government had decided to appeal against yesterday's decision by the environment ministry to give the project the go-ahead.
He said he had called the first "technical talks" between the regional government and affected mayors to discuss a pending appeal to the Constitutional Court.
Emiliano said he deplored the clashes, saying "the government has proved incapable of listening to Puglia".
"Using the massive deployment of forces that was set up today, the government shows its incapacity to listen and politically process the requests of an entire region which has in its ruling program, elaborated from the bottom and voted for by hundreds of thousands of Puglians, the transfer of the TAP link to another area," he added.
Anti-establishment 5-Star Movement (M5S) leader Beppe Grillo attacked Emiliano for allegedly seeking to boost his campaign to become leader of the ruling centre-left Democratic Party (PD), in a race where Emiliano is lagging far behind favourite and former PD leader Matteo Renzi.
"He should wake up or resign, not try to be a showman," Grillo said.
Emiliano announced earlier this month that uprooting olive trees to make way for the TAP was "illegal".
Voicing his support for farmers who clashed with police, Emiliano, however, added that "the regional government does not have instruments to stop a project that the government has told police to protect, an operation considered absolutely strategic".
Before he became Italian premier, then Foreign Minister Paolo Gentiloni said last year that the creation of the pipeline is necessary for the diversification of provision sources, not just for the EU and the Balkans but also for Italy.
Speaking in June at the foreign ministry in Rome, Gentiloni said that he hopes to see Azeri gas reach Italy by 2020.
The objective, recalled Gentiloni, is to boost economic and political relations between Rome and Baku, which he considered a "strategic regional partner".
The TAP consortium is made up of the following companies: BP (20 per cent), SOCAR (20 per cent), Snam (20 per cent), Fluxys (19 per cent), Enags (16 per cent) and Axpo (5 per cent).
It is set to invest 5.6 billion euros in the gas pipeline in the next few years.
With ANSA
