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Over the past few weeks many tents and flags have transformed the green into what protesters have called Democracy Village.
They include anti-war protester Brian Haw, who has previously won legal battles to be allowed to continue his demonstration outside Parliament.
Boris Johnson has now decided to begin trespass proceedings at the High Court.
Protesters are expected to attend court to fight the injunction.
In 2007 it was made illegal to hold an unauthorised protest within a square mile of Parliament.
A spokesman for the mayor said: "Parliament Square is a world heritage site [OP note: No it isn't] and top tourist attraction that is visited by thousands of people and broadcast around the world each day.
"The mayor respects the right to demonstrate, however the scale and impact of the protest is now doing considerable damage to the square and preventing its peaceful use by other Londoners, including those who may wish to have an authorised protest."
One protester, Chris Knight, said: "I think it is an absolute scandal, it's a disgrace.
"He [Boris Johnson] should be supporting democracy, not opposing it.
"If Boris wins this case it means freedom of speech, freedom of assembly will be put in the dustbin.
"I'd rather he was put in the dustbin."
The move has been welcomed by Westminster City Council whose leader, Councillor Colin Barrow, has accused the protesters of "hijacking" the square.
Source: BBC (and jslayeruk for the proof the spokesman doesn't know what he's talking about)