The micro-bogging site apparently on its own took the action against the Jamaat-ud-Dawa (JuD) chief.
The site can suspend user accounts for violation of rules which include impersonation, copyright infringement, violence and threats.
Saeed's Twitter account @HafizSaeedJUD now reads "account suspended".
Some media reports said Saeed had created two new accounts to become active on Twitter.
His spokesman or anyone from JuD was not immediately available for comment.
With logistical support from the Pakistan government, Saeed on Friday told his supporters that Pakistanis should come forward and help the Kashmiris in getting "freedom" from India.
"If India can send troops to Afghanistan to help the US, then Mujahideen have every right to go to Kashmir and help their brethren. Kashmiris are clamouring for help and it is our duty to respond to their call," he had said addressing a JuD congregation at historical Minar-e-Pakistan ground in Lahore.
The United Nations declared JuD a terrorist organisation and also individually designated Saeed as a terrorist in December 2008.
Saeed, who also has a US bounty of 10 million dollars on him, roams freely in Pakistan and often addresses public rallies in which he routinely makes inflammatory statements against India.
Pakistan has said that there is no case against Saeed and that he is free to move in the country as a Pakistani national.
India has repeatedly demanded Pakistan to hand over Saeed for questioning over his role in the 2008 Mumbai attacks that claimed 166 lives.
JuD was banned in 2002 by Pakistan but later a court lifted the ban due to lack of evidence on the group's involvement in militancy.
The group is also banned by the US, the EU, India and Russia as a terror organisation.
In June 2014, the US declared JuD a subsidiary of terror group Lashkar-e-Taiba.