Tunnelling is set to start for a line from Turin to Lyon, which is expected to cut the travel time by nearly half.
Local residents built barricades to prevent heavy machinery from starting work in the picturesque Val di Susa, in northern Italy.
Police used fire hoses and tear gas to disperse them.
Police had to escort the contractor's bulldozers to the high Alpine site where work is due to begin.
Demonstrators set on fire barricades erected to keep out intruders, but failed to halt the works.
Despite environmental objections, the Italian government is determined to go ahead with the project, which is heavily backed by EU money and is estimated to cost 15bn euros (?13bn).
Work is due to start by 30 June or the country risks losing the hundreds of million euros the EU is contributing, a minister said at the weekend.
Earlier, more than 3,000 people took part in a peaceful, overnight candle-lit prayer vigil against the project.
"The majority of residents of the valley are against this project which damages the environment and is an absurdity for public finances," said Paolo Ferrero, secretary of Italy's Communist Refoundation Party.
Residents fear that, despite safeguards built into the multi-billion euro project in a deal signed in 2001, an area of outstanding natural beauty is going to be spoilt.
Other protests have been announced on the internet, with a demonstration due in Rome on Tuesday, reports say.
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